Green Tea Leaves and Rosemary Extracts Selectively Induce Cell Death in Triple-Negative Breast Cancer Cells and Cancer Stem Cells and Enhance the Efficacy of Common Chemotherapeutics

While incredible medical advancements in chemotherapeutics development for cancer treatment have been made, the majority of these are not selective in their mechanism of action, leading to adverse efects. Given the systemic toxicity associated with these therapies, they arenotwellsuitedforlong-termuse.Naturalhealthproducts,orNHPs,mayprovideawaytoselectivelytargettheoxidativeandmetabolic vulnerabilitiesincancercells.Whitetea( Camelia sinensis ) and rosemary ( Salvia rosmarinus ) are two natural extracts that have been studiedextensivelyfortheirmedicinalproperties.However,theiranticanceractivityandmechanismofactionareyettobefullyelucidated. Wehaveexaminedtheextracts’cancercell-killingabilityaswellastheirinteractionswithcommonchemotherapeuticsinMDA-MB-231cells,atriple-negativebreastcancercellline, in vitro . Cell death measurement, morphological and biochemical characterization of apoptotic cell death, mechanisms of action (mitochondrial depolarization and oxidative stress), and immunofuorescence assays to estimate the percentage of cancer stem cells (CSCs) were performed following treatment with Synthite tea extract (STE) and rosemary extract (RE),providedby SynthiteIndustriesLimitedalone andincombinationwithcisplatinandpaclitaxel.Te keyfndings inthis study are that STE and RE alone demonstrated very efcient anticancer activity against TNBC, and more importantly, the administration of the extracts in conjunction with cisplatin and paclitaxel sensitizes cancer cells to achieve enhanced cell death. In addition, CSCs were found to be sensitive to treatment with STE alone and in combination with RE and exhibited greater sensitivity to combination therapies compared to chemotherapeutic alone. Te signifcance of these observations is that STE and RE, well-tolerated NHPs, have the potential to enhance the efcacy of current chemotherapeutics when combined, as well as prevent relapse for TNBC.


Introduction
Breast cancer is the most common cancer among women worldwide, making up a quarter of all cancer diagnoses [1].Hormones play an important role in cancer's progression and, as such, present important targets for combatting the disease [2].Breast cancer can generally be split into three subtypes: (i) receptor-positive tumours (estrogen and progesterone receptors), (ii) tumours with high levels of HER2 protein, and (iii) receptor-negative tumours with low HER2 production [2].Breast cancers characterized by the lack of hormone receptors have a much poorer prognosis [2].Belonging to the third subgroup, triple-negative breast cancer accounts for around one-tenth [3], or 10 to 15 percent, of all breast cancers [2].It is named after its three defciencies such as low HER2 production and negative estrogen and progesterone receptors [2].Te type of treatment administered is heavily infuenced by hormone and protein production as they may be used as targets [2].Lack of molecular targets makes TNBC an aggressive and challenging cancer to treat.Terefore, it is important to develop more efective treatment options [2].
While the advancements in medical technologies have resulted in improved healthcare outcomes for cancer patients, they face considerable limitations as the disease progresses [4].With regard to preventative measures, breast cancer can be detected in its early stages by using screening methods [4].At these stages, surgical methods may also be used to avoid further development [4].Generally, tumourectomy is associated with high survival rates [5].However, surgical methods risk aggravating the disease as the tumour can disseminate, advancing cancer to a late metastatic stage where surgical options are limited or futile [5].Currently, chemotherapeutic regimens are the standard treatment plan once the disease has progressed to its later stages.Breast cancer is typically treated with cisplatin or paclitaxel, which are platinum-based and taxane-based drugs, respectively [6,7].While chemotherapeutics are an incredible medical advancement, they are not selective in their mechanism of action and may additionally target noncancerous cells [8,9].For example, paclitaxel is known to target the mitotic spindle assembly and cell division, which are the traits shared by both cancerous and healthy cells [8,9].
Cancer stem cells (CSCs) are a self-renewing population of cells within a tumour [10][11][12].CSCs have the potential to self-renew indefnitely due to the dysregulation of signalling pathways regulating self-renewal mechanisms [11].In this manner, the proliferation of CSCs drives tumourigenesis or tumour formation [11].Genotoxic agents such as cisplatin induce apoptosis in cancer cells by damaging DNA and triggering the DNA damage response [10].CSCs have been found to promote DNA repair following DNA damage [13], thereby repopulating tumour masses after treatment with genotoxic chemotherapeutics [10].CSCs have also been observed to exhibit chemotherapeutic resistance: diferential survival of CSCs following treatment with chemotherapy has been found to increase the percentage of CSCs in progeny, subsequently leading to the formation of a chemoresistant tumour [14,15].As such, CSCs are thought to risk failure of anticancer chemotherapeutics and relapse in patients [10].Current anticancer treatments target the entire population of cells within a tumour, but the proliferation potential of most tumour cells is limited [11].Treatments that do not specifcally target CSCs may shrink a tumour and later lead to tumour regrowth if a sufcient number of CSCs survive and proliferate indefnitely [11].Terapies that specifcally kill CSCs may deprive tumours of the ability to regenerate cells and grow [11,16].
TNBCs seem enriched with CSCs compared to other breast cancer subtypes [17].As chemotherapy resistance is frequently developed in TNBC patients [18] and around 40% of TNBC patients relapse after treatment with current chemotherapeutics [19], treatments targeting CSCs may improve TNBC patient's prognosis by attaining a stable tumour remission [10,14].
CSCs are often identifed by the presence of certain surface molecules [12,20].CSCs in TNBCs are associated with the phenotypes CD44+, CD24−, and ALDH1+ [21][22][23].CD44+ and CD24− CSCs are thought to be highly invasive, contributing to tumourigenesis and metastasis [21,23,24].In a study by Al-Hajj et al. [25], only 100 CSCs with the CD44+ and CD24− phenotype were needed to initiate tumour formation in mice, while tens of thousands of cells with other phenotypes were unable to form tumours. CD44 is a glycoprotein adhesion molecule [20] and is highly expressed in the MDA-MB-231 cell line [23,24,26].Tis study will analyze the expression of the CD44 cell surface marker to investigate the ability of STE and RE, administered alone and in combination with current chemotherapeutics, to induce apoptosis in MDA-MB-231 CSCs.
Te lack of selectivity displayed by chemotherapies may cause adverse side efects that ultimately lead to patient's death [27].As such, a patient is limited to the number of cycles of chemotherapy they may take, as determined by the maximum lifetime dose [27].In addition, many cancers are known to develop resistance to chemotherapeutic drugs, thus reducing their efectiveness [28].Given the systemic toxicity and the risk of resistance associated with these treatments, they are not suitable as long-term treatments [27,28].
Certain biochemical vulnerabilities can be exploited to target cancer cells selectively [29,30].Due to their hyperactive metabolic activity, cancer cells generally display increased intracellular levels of reactive oxygen species (ROS) [29].ROS are highly reactive, unstable oxygen-containing molecules that may interact with other molecules in the cells [29].While their occurrence is important for healthy cellular processes, an excess of ROS may damage the DNA, RNA, and proteins [29].Naturally, there are protective cellular mechanisms tasked with managing the balance of ROS [29,30].However, cells will undergo apoptosis once the levels of oxidative stress are pushed beyond the level by which the body may cope [30].Previous works have shown that various NHPs can exploit these oxidative weaknesses to selectively induce apoptosis without harming normal cells [31].Furthermore, the diferences between cancerous and noncancerous cell mitochondrial activity may also be exploited [30].Te Warburg efect explains that cancerous cells prefer metabolism via glycolysis which leads to a buildup of lactic acid within the cytosol [32].An acidic cytosol is one of the multiple factors leading to the hyperpolarization of cancerous mitochondria [33].Terapeutics that target the mitochondrial membrane potential (MMP) do so by initiating an internal stress stimulus which causes proapoptotic proteins to translocate to and permeabilize the outer mitochondrial membrane [34].Once permeabilized, the mitochondria become depolarized and apoptogenic factors from the intermembrane space are released into the cytosol of the cell, ultimately leading to apoptosis [34].
Natural health products (NHPs) or botanical substances have served older human civilizations for a wide range of medicinal purposes [35].Over the course of hundreds of years, the ancient Chinese, Ayurvedic, and First Nation tribes used plant-based medicines to treat acute and chronic diseases [35,36].Teir long history of medicinal use has warranted further investigation by researchers across the world [36].For example, turmeric, a plant native to southeastern Asia, has been extensively shown to contain phytochemicals with antioxidant, anti-infammatory, antimutagenic, antimicrobial, and anticancer properties.Pertaining to the oncological feld, isolated compounds from NHPs account for three-quarters of current chemotherapeutic drugs [31].

2
Evidence-Based Complementary and Alternative Medicine As their mechanisms have not fully been elucidated, medical professionals are rightfully skeptical of their efcacy in a clinical setting [35,36].Te skepticism surrounding NHPs is further amplifed by the lack of information on their interactions with common chemotherapeutic poses [31].Along with their therapeutic value, NHPs are known to be safe for consumption in humans with minimal risk and thus are a promising candidate for further research on their anticancer activities [31].
Camellia sinensis, a plant native to China, belongs to the tea plant family Teaceae [37].Te leaves of C. sinensis are used to prepare a variety of teas, for example, green, black, oolong, and white tea [37].Te plant contains a multitude of bioactive compounds such as polyphenols, favonoids, and vitamins [37].As such, these teas have been linked with therapeutic properties against cardiovascular diseases, cancers, and infammatory diseases [37].Previous studies have demonstrated the anticancer activity of Camellia sinensis [38].Te extract has been shown to selectively target MDA-MB-231 cells, thereby reducing cell viability in the TNBC cells while having no toxic efect on noncancerous cells [38].Te extract was also found to reduce cell migration in MDA-MB-231 by 50%, which has implications for addressing tumour progression [38].
Synthite tea extract (STE) is an extract prepared from fresh leaves of Camellia sinensis by using a proprietary procedure described by Synthite Industries Ltd. Te minimalistic preparation results in a high retention of polyphenol antioxidants such as epicatechins [39].Tese phytochemicals are known to interact with and quench ROS in vitro [39].As explained in an earlier section, ROS is involved in the pathology of several diseases [29], as such the epicatechins in STE may have therapeutic value [39].Specifcally, epigallocatechin-3-gallate has been shown to have apoptosisinducing and antiproliferative activity against diferent cancers [40].
Salvia rosmarinus, or more commonly known as rosemary, is a shrub-like plant that is native to the Mediterranean region [41].Te plant is characterized by its evergreen and needle-shaped leaves, and it belongs to the mint family Lamiaceae [41].Aside from being a commonly used spice, the plant has long been used in medicinal settings due to its anti-infammatory, antioxidant, antimicrobial, antiproliferative, and antitumour activities [41].Among other bioactive compounds, RE contains phenolic diterpenes such as carnosol, carnosic acid, and rosmarinic acid [42].
Rosemary has been proven to display powerful anticancer efcacy in numerous cancers in vitro, such as cancer of the lung, prostate, liver, and breast [42].Carnosic acid appears to be the plant's primary anticancer component, as it was found to exhibit antiproliferative efects in cancer cells [41].As well, rosmarinic acid had a signifcant anticancer activity against several cell lines [42].In MDA-MB-231 cells, the extract was found to exhibit an antiproliferative efect by inhibiting cell growth in a dosedependent manner [43].Previous studies have also shown that the extract exhibits an antimigratory efect on MDA-MB-231 cells by reducing cell motility [43].Previous studies thus demonstrate rosemary extract's potential as an anticancer treatment [43], and so its mechanisms of action as well as its interactions with common chemotherapeutics should be investigated.
TNBCs were chosen for this study due to their difculty to treat and aggressivity, which creates a need to develop safer and more efective therapy.Compounds from Camellia sinensis and Salvia rosmarinus have been shown to exhibit anticancer activity in the past [38,43].Te advantage of using the total extract is that they are well-tolerated, while purifed compounds may be toxic.Furthermore, total extracts contain multiple compounds which may have synergistic efects on anticancer activity [36].We have previously shown that STE was efective in inducing apoptosis in lymphoma cells [44].However, the efcacy of STE and RE alone has not been studied in detail in MDA-MB-231 cells.Furthermore, STE and RE may sensitize TNBC cells to chemotherapeutics, leading to a more efective treatment of this aggressive cancer.Patients are also limited in the number of chemotherapeutic cycles they may undergo [27], while STE and RE are well-tolerated and can be administered long-term.Tis study aims to investigate the anticancer efects of STE and RE in MDA-MB-231, a TNBC cell line, as well as their interactions with common chemotherapeutics.We hypothesize that the multiple phytochemicals in these well-tolerated extracts have their own anticancer efects and enhance the efcacy of common chemotherapeutics if administered together.

Synthite Tea and Rosemary Extraction and Preparation.
Both extracts STE and RE used in this project are extracted and prepared by Synthite Industries Private Ltd., a privately held company based in Kochi, India.Synthite obtains these materials from its own tea and rosemary forms and produces these extracts on a large scale.Details pertaining to the extraction and preparation of STE and RE are owned by Synthite Industries and are not publicly disclosable.Synthite has provided HPLC analysis profles and certifcate of analysis (COA) for both extracts.Tis information is provided in Supplemental fles.Tese extracts have been approved for human consumption in India as supplements.
A fne powder of Synthite tea was dissolved in distilled water to make an aqueous extract.Rosemary, in a CO 2 supercritical fuid form, was dissolved in DMSO.
To assess the efcacy of both extracts in our cell culture models, cells were plated and grown to 50-70% confuence prior to treatment with STE or RE, at increasing concentrations ranging from 0.01 mg/mL to 0.25 mg/mL.After treatment, cells were analyzed for the efcacy of STE and RE, as described below.All cells were cultured for ≤4 months, before being discarded, and fresh frozen cells were used to continue studies, lasting longer than the 4 months period.

Assessment of Programmed Cell Death Induction.
Annexin V binding assay and propidium iodide staining were performed to, respectively, monitor early apoptosis and cell permeabilization, a marker of necrotic or late apoptotic cell death.Cells were washed with phosphate bufer saline (PBS) and suspended in Annexin V binding bufer (10 mM of HEPES, 140 mM of NaCl, 2.5 mM of CaCl2, and pH of 7.4) with green fuorescent Annexin V Alexa Fluor 488 (1 : 20) (Life Technologies Inc, Cat.no.A13201, Burlington, ON, Canada) and 0.01 mg/mL of red fuorescent PI (Life Technologies Inc, Cat.no.P3566, Burlington, ON, Canada) for 15 minutes at 37 °C and protected from light.Te percentage of early (green) and late apoptotic cells (green and red) and necrotic cells (red) was quantifed with a Tali Image-Based Cytometer (Life Technologies Inc., Cat. no.T10796, Burlington, ON, Canada).Cells from at least 13 random felds were analyzed by using both the green (ex.458 nm; em.525/20 nm) and red (ex.530 nm; em.585 nm) channels.Fluorescent micrographs were taken at 200x or 400x magnifcation using LAS AF6000 software with a Leica DMI6000 fuorescent microscope (Wetzlar, Germany).Cells monitored with microscopy were counterstained with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) with a fnal concentration of 10 μM following the 15-minute incubation.Te protocol used is similar to that of previously published work [31].

Assessment of Cellular Activity and Viability.
To examine the viability of breast cancer cells after treatment, cells were incubated for 48 hours following the desired treatments and then incubated once more with cell proliferation reagent WST-1 (Catalogue no.05 015 944 001, Roche Diagnostics) for 3 hours at 37 °C.Absorbance readings of the formazan product were obtained at 450 nm by using a spectrofuorometer (SpectraMax Gemini XS, Molecular Devices, Sunnyvale, CA).Viability readings were analyzed using GraphPad Prism 6.0 software and expressed as a percentage of the control untreated groups.

Evaluation of Mitochondrial Membrane Potential.
0.1 μM of tetramethylrhodamine methyl ester (TMRM; Gibco BRL; VWR; Cat.no.89139-392) was used for detecting mitochondrial membrane potential (MMP), an indicator of healthy intact mitochondria.Following incubation with TMRM, cells were collected, washed with 1 × PBS, resuspended in PBS, and then analyzed using the Tali Image-Based Cytometer (Life Technologies Inc; Cat.no.T10796).Cells from 13 random felds were analyzed by using the red (ex � 530 nm; em � 585 nm) channel.Fluorescent micrographs were taken at 200x or 400x magnifcation by using LAS AF6000 software with a Leica DMI6000 fuorescent microscope (Wetzlar, Germany).Cells monitored with microscopy were stained with 0.1 μM of TMRM and counterstained with 10 μM of Hoechst 33342 (Molecular Probes, Eugene, OR, USA).Te protocol used is similar to that of the previously published work [31].
Cells were treated for the indicated durations, collected, centrifuged at 3500 × g for 5 min, and resuspended in PBS.Te percentage of DCF-positive cells was quantifed by using the Tali Image-Based Cytometer (Life Technologies Inc., Burlington, ON, CA, Cat no.T10796) using 13 random felds per group with the green channel (excitation: 458 nm; emission: 525/20 nm).
N-acetylcysteine (NAC) was used to evaluate the efect of ROS inhibition on the apoptosis induction of the extracts.Cells were treated with varying concentrations of both extracts with or without NAC (5 mM per well), a known inhibitor of ROS, and then incubated for 48 hours.Te cell viability was assessed by using the WST-1 viability assay as described in Section 2.4.
Fluorescent micrographs were taken at 200x or 400x magnifcation using LAS AF6000 software with a Leica DMI6000 fuorescent microscope (Wetzlar, Germany).Cells monitored with microscopy were stained with 20 μM of H2DCFDA and counterstained with 10 μM of Hoechst 33342 (Molecular Probes, Eugene, OR, USA).Te protocol used is similar to that of previously published work [31].

Assessment of Cell Viability by Analyzing Cell Morphology.
Brightfeld images were taken at 200x or 400x magnifcation by using ToupLite software with the OMAX A35100U Microscope.Te images were used to monitor treatment groups for apoptotic features, such as cell shrinkage, membrane blebbing, and nuclear condensation.Slides were preserved with 50% glycerol (Sigma-Aldrich, Germany), and images were visualized using LAS AF6000 software with a Leica DMI6000 fuorescent microscope (Wetzlar, Germany).Te protocol used is similar to that of previously published work [23].

Statistical Analysis.
Statistical analyses of wound healing assays and immunofuorescence staining for cancer stem cells were performed in R (R Core Team, 2022) by using a one-way analysis of variance (ANOVA) and Tukey's multiple comparison of means.All other statistical analyses were performed by using a two-way ANOVA in GraphPad Prism version 6.0 for Windows, GraphPad Software, San Diego, California, USA (https://www.graphpad.com).All trials were conducted for at least three independent times.

Synthite Tea and Rosemary Extracts Reduce Viability of
Triple-Negative Breast Cancer Cells.Tis work initially assessed the anticancer activity of each extract as a standalone therapeutic against MDA-MB-231 breast cancer cells.To determine the apoptosis-inducing capabilities of both STE and RE, an Annexin V and propidium iodide (AVPI) assay was used.Te cells were stained with the fuorescent apoptotic-marking dyes, Annexin V (AV) and propidium iodide (PI).AV binds to phosphatidylserine once it has externalized onto the outer membrane, a hallmark of apoptosis.Meanwhile, PI is a DNA intercalating dye that will only permeabilize with apoptotic cells.Te absence of AV or PI staining represents viable cells, as shown in the graph legend.On the left of Figures 1(a), 1(b), 2(a), and 2(b), the percentage of apoptotic cells is graphed.Concentrations ranging from 0.10 mg/mL to 0.25 mg/ mL of aqueous STE were tested at 24 (Figure 1(a)) and 48 hours (Figure 1(b)).At 24 hours, concentrations of 0.15 mg/mL of STE and onward signifcantly induced apoptosis when compared to the negative control.At 48 hours, every tested concentration of STE showed statistically signifcant apoptosis induction.Terefore, the extract displays clear time and dose dependency.
An analysis of cell morphology confrms these results at 24 (Figure 1(a)) and 48 hours (Figure 1(b)).Te control group is characterized by long, spindle-like shapes and the absence of apoptotic features, such as cell shrinkage, membrane blebbing, and nuclear condensation.However, as the concentration of the extract increased, the cells distinctly difered with the control group's morphology.STE treatment groups are characterized by the previously mentioned apoptotic markers, most evidently in the 0.25 mg/mL group.
Similarly, concentrations of 0.01 mg/mL-0.10mg/mL of RE were tested at 24 (Figure 2(a)) and 48 hours (Figure 2(b)).At 24 hours, 0.01 mg/mL did not induce signifcant apoptosis, but 0.025 mg/mL and onwards did.A similar trend was observed at 48 hours, where all concentrations aside from 0.01 mg/mL induced apoptosis.An analysis of cell morphology (Figures 2(a) and 2(b)) may confrm these results.Te RE treatment groups clearly displayed apoptotic morphology.Interestingly, the 0.025 mg/mL treatment group induced less apoptosis at 48 hours than at 24 hours, suggesting that its efcacy diminished over time.
Fluorescent microscopy (Figures 1(c) and 2(c)) served as supplementary qualitative evidence in support of the AVPI assays.Cells were once again stained with AV (green) and PI (red) prior to treatment with STE and then photographed at 48 hours.In addition, cells were counterstained with Hoechst (blue).All cells, viable or apoptotic, stained blue, while only apoptotic cells stained red and/or green.As shown by the increase in red and green fuorescence, STE and RE signifcantly induced apoptosis in triple-negative breast cancer with dose dependency (Figures 1(c) and 2(c), respectively).Evidence-Based Complementary and Alternative Medicine

Investigating STE and RE's Interactions with Common
Chemotherapeutics: Herb-Herb and Herb-Drug Interactions.
According to Figures 3(a) and 3(d), STE and RE are signifcantly much more efective when administered in combination.Results from the AVPI assay (Figure 4(a)) suggest a synergistic efect, as the combination treatment reduced cell viability more than either extract did on its own.Te brightfeld images confrm these fndings, as the cells have signifcantly shrunk and condensed.
As shown in Figures 3(b) and 3(c), Synthite tea positively interacted with cisplatin and Taxol.Combining relatively low doses of both STE and cisplatin resulted in a great reduction in cell viability when compared to either component on its own.As well, STE enhanced Taxol's anticancer activity.Te STE-Taxol combination group induced more apoptosis than Taxol did on its own but was not found to be statistically diferent than STE on its own.Provided on the right in fgures Figures 4(b) and 4(4(c), there is a distinct loss in healthy cellular morphology in the STE-drug combination groups.
On the other hand, rosemary did not interact in any manner with either chemotherapy.Te RE-cisplatin group did not result in more cell death than RE or cisplatin did on their own.In the same vein, the RE-Taxol group was also not found to have better anticancer activity than either RE or Taxol did on their own.Te brightfeld images (Figures 3(b) and 3(c)) only partially agree with AVPI results.Contrary to the AVPI graph, there is a signifcant increase in apoptotic features in the cisplatin-RE group when compared to cisplatin on its own.Moving on, the Taxol-RE image shows no discernible diference between Taxol and Taxol-RE, as expected.
Finally, the herb-herb-drug groups induced signifcant cell death in breast cancer.In Figures B and D, we see that cisplatin-STE-RE was much more efective than the cisplatin-STE and the cisplatin-RE groups were.As well, the Taxol-STE-RE group induced more cell death than the STE-Taxol and RE-Taxol groups.Te micrographs on the right show that in both herb-herb-drug groups, the cells have lost all resemblance to the control group.Most importantly, as shown in Figure 4(d), neither of the two STE-REchemotherapy groups was more efective than the STE-RE group.
Fluorescent images of STE, RE, cisplatin, Taxol, and all their combinations are provided in Figure 5.As the chemotherapeutics were administered at low doses, they did not induce much apoptosis, hence the low amount of red and green fuorescence.However, as the chemotherapies were supplemented with STE or RE, there appears to be more cell death.Finally, when the chemotherapies were combined with both the extracts, almost every single cell underwent PCD as they were stained in either red or green.Te fuorescent micrographs are in accordance with the results in Te statistical signifcance of cell viability was analyzed for every combination therapy and its individual components.Compared to the control, cell viability was signifcantly reduced in cells treated with 0.10 mg/mL of STE (p < 0.0001), 0.025 mg/mL of RE (p < 0.05), and a combination of STE + RE (p < 0.0001).Treatment with 0.10 mg/mL of STE + 0.025 mg/mL of RE signifcantly reduced cell viability compared to treatment with either extract alone (p < 0.00001 for both comparisons).
Cell viability was signifcantly reduced compared to the control in cells treated with 0.50 µM of cisplatin (p < 0.01), cisplatin combined with 0.10 mg/mL of STE (p < 0.0001), cisplatin combined with 0.025 mg/mL of RE (p < 0.01), and cisplatin combined with both extracts (p < 0.0001).Compared to cells treated with cisplatin alone, cell viability was signifcantly reduced in cells treated with cisplatin + STE (p < 0.00001) and cisplatin + STE + RE (p < 0.00001).Tere was no signifcant diference in cell viability in cells treated with cisplatin + RE compared to cells treated with cisplatin alone and cells treated with RE alone.Cell viability was signifcantly reduced in cells treated with cisplatin + STE + RE compared to cells treated with cisplatin alone (p < 0.00001), STE alone (p < 0.00001), and RE alone (p < 0.00001), STE + cisplatin (p < 0.00001), and RE + cisplatin (p < 0.00001).Tere was no signifcant diference in cell viability between cells treated with cisplatin + STE + RE and cells treated with STE + RE.
Cell viability was signifcantly reduced compared to the control in cells treated with 0.01 µM of Taxol (p < 0.05), Taxol combined with 0.10 mg/mL of STE (p < 0.0001), Taxol combined with 0.025 mg/mL of RE (p < 0.05), and Taxol combined with both extracts (p < 0.0001).Compared to cells treated with Taxol alone, cell viability was signifcantly reduced in cells treated with Taxol + STE (p < 0.00001).Tere was no signifcant diference in cell viability in cells treated with STE + Taxol compared to cells treated with STE alone.Tere was no signifcant diference in cell viability in cells treated with Taxol + RE compared to cells treated with Taxol alone and cells treated with RE alone.Cell viability was signifcantly reduced in cells treated with Taxol + STE + RE compared to cells treated with Taxol alone (p < 0.00001), STE alone (p < 0.00001), and RE alone (p < 0.00001), STE + Taxol (p < 0.01), and RE + Taxol (p < 0.00001).Tere was no signifcant diference in cell viability between cells treated with Taxol + STE + RE and cells treated with STE + RE.None of the tested concentrations were signifcantly different compared to the control, and so none of the administered concentrations induced apoptosis in noncancerous cells.Terefore, we have demonstrated that both NHPs induce cancer-specifc apoptosis, leaving normal cells unafected.

Assessment of Anticancer Activity of STE, RE, Chemotherapeutics, and Combinations with Wound Healing Assay.
Compared to the untreated control, wound closure distance was signifcantly greater in cells treated with 0.1 mg/mL of STE alone (p < 0.01), 0.1 mg/mL of RE alone (p < 0.05), and 0.1 mg/mL of STE and 0.1 mg/mL of RE combined (p < 0.01) (Figure 6(a)).
Te average wound closure distance following treatment with cisplatin and cisplatin-extract combinations is shown in Figure 6(b).Wound closure distance in cells treated with cisplatin alone was not signifcantly diferent than that of the negative control.Compared to the untreated control, wound closure distance was signifcantly greater in cells treated with 0.1 mg/mL of STE and 0.50 μM of cisplatin combined (p < 0.05) and 0.1 mg/mL of RE and 0.50 μM of cisplatin combined (p < 0.001), as well as 0.1 mg/mL STE, 0.1 mg/mL RE, and 0.50 μM cisplatin combined (p < 0.0001).Wound closure distance was signifcantly greater in cells treated with the RE-cisplatin combination (p < 0.01) and the STE-RE-Taxol combination (p < 0.0001) compared to cells treated with cisplatin alone.
Te average wound closure distance following treatment with Taxol and Taxol-extract combinations is shown in Figure 6(c).Treatment with 0.01 μM of Taxol alone (p < 0.01), 0.1 mg/mL of STE and 0.01 μM of Taxol combined (p < 0.0001), and 0.1 mg/mL of RE and 0.01 μM of Taxol combined (p < 0.01), as well as 0.1 mg/mL of STE, 0.1 mg/mL of RE, and 0.01 μM of Taxol combined (p < 0.0001) significantly increased the wound closure distance compared to the untreated control.Wound closure distance was significantly greater in cells treated with the STE-Taxol combination (p < 0.0001) and the STE-RE-Taxol combination (p < 0.01) compared to cells treated with Taxol alone.
Average wound closure distance was signifcantly greater in cells treated with STE alone compared to cells treated with Taxol alone (p < 0.05) and compared to cells treated with cisplatin alone (p < 0.01).Tere was no signifcant diference in wound closure distance in cells treated with RE and cells treated with either chemotherapeutic alone.Wound closure distance in cells treated with the STE-RE combination was signifcantly greater than that of the cells treated with cisplatin alone (p < 0.01) but did not difer from Taxol alone.

Te Induction of ROS Production in Breast Cancer.
Te introduction outlines the importance of ROS as cancerspecifc targets for NHPs.Te H2DCFDA assay (Figure 7  Evidence-Based Complementary and Alternative Medicine 0.05 mg/mL of rosemary extract induced the production of ROS in MDA-MB-231 cells.In contrast, Synthite tea extract displayed minimal ROS production and was not observed to be statistically diferent than the negative control group.Te RE-chemo groups induced the same levels of ROS as the RE standalone treatment.Terefore, there are neither enhancing nor inhibitory efects on the extract's ROS production when combined with standard chemotherapeutics.Evidence-Based Complementary and Alternative Medicine However, when RE was combined with STE, its ROSinducing activity was no longer observed.Fluorescent microscopy (Figure 7(b)) confrms the results found in Figure 7(a).Green fuorescence indicates the production of ROS, while all cells will stain blue.On the top is the blue channel on its own, while the bottom is the green channel on its own.When compared with the control group, the rosemary treatment groups have much more green fuorescence.Meanwhile, the STE treatment showed minimal to no green fuorescence.Terefore, RE induces ROS in breast cancer while STE does not.

Te Dependence on Oxidative Stress to Induce Apoptosis.
As outlined in Section 3.3, only one of the extracts, rosemary, was found to induce ROS in breast cancer.In this section, we investigate the efect that ROS has on rosemary's ability to induce apoptosis.Cells were pretreated with Nacetylcysteine (NAC), a known inhibitor of oxidative stress.Ten, cells were treated with either STE or RE at concentrations ranging from 0.01 mg/mL to 0.25 mg/mL.As shown in Figure 8(a), the addition of NAC signifcantly inhibited RE's apoptosis induction in breast cancer.Te majority of RE concentrations (0.025 mg/mL-0.15mg/mL) were afected by the addition of NAC, while the highest doses were not.In contrast, STE's anticancer activity was completely unafected by NAC (Figure 8(b)).Tese fndings were confrmed with fuorescent microscopy (Figures 9(a) and 9(b)).In the micrographs of 0.10 mg/mL of RE, there is signifcant red and green fuorescence, indicating interactions with Annexin V and PI apoptotic markers.Yet, when NAC is added, there is a minimal green or red fuorescence.Furthermore, the micrographs in Figure 8(b) show that the red and green fuorescence displayed by the STE treatment group is similar to that of the STE supplemented with NAC.As is consistent with the results in Section 3.3, RE is dependent on the induction of ROS to induce apoptosis in breast cancer, while STE is not.Evidence-Based Complementary and Alternative Medicine depolarization.Tetramethylrhodamine methyl ester (TMRM) served to quantify mitochondrial stability.TMRM will bind with healthy mitochondria; therefore, high levels of red fuorescence indicate intact and stable mitochondria.Cells were pretreated with TMRM and then treated with STE, RE, Taxol, cisplatin, and their combinations and were assessed at 48 hours.Rosemary, cisplatin, and Taxol were unable to afect MMP.In contrast, every treatment group involving STE completely collapsed the MMP to almost 0. Synthite tea showed signifcant destabilizing efects on cancerous mitochondria and was not inhibited by RE or either chemotherapy.Furthermore, while RE did not afect the MMP on its own, both the RE-cisplatin and RE-Taxol groups depolarized the mitochondria (Figure 9(a)).

Evidence-Based Complementary and Alternative Medicine
To provide supplementary evidence, this experiment was not repeated but instead analyzed by using fuorescent microscopy (Figure 9(b)).Red fuorescence is indicative of healthy mitochondria, while its absence indicates mitochondrial depolarization.Hoechst will bind with every cell and is shown by the blue fuorescence.Each treatment group is accompanied by two side-by-side images.On the left is a merged image of both the red and blue fuorescent channels, while on the right is the red channel on its own.Te control is characterized by signifcant red fuorescence, while every STE group had minimal red fuorescence if any at all.Te RE group is comparable with the control.Terefore, the microscopy is in accordance with quantitative data, where STE acts by targeting the MMP, while RE does not.

Sensitivity of Cancer Stem Cells to STE, RE, Chemotherapeutics, and Combinations.
Compared to untreated cells, the average percentage of cells expressing CD44 was signifcantly less in cells treated with 0.25 mg/mL of STE (p < 0.001) and in cells treated with a combination of 0.1 mg/ mL of RE and 0.25 mg/mL of STE (p < 0.01) (Figure 10(a)).Te percentage of cells expressing CD44 in cells treated with 0.1 mg/mL of RE was not signifcantly diferent than the percentage in the negative control.Tere were signifcantly less cells expressing CD44 in cells treated with STE and RE compared to cells treated with RE alone (p < 0.05).Te percentage of cells expressing CD44 was signifcantly less in cells treated with STE compared to cells treated with a combination of RE and STE (p < 0.05).
Figure 10(b) illustrates the average percentage of cells expressing CD44 following treatment with cisplatin and cisplatin-extract combinations.Compared to untreated cells, the percentage of cells expressing CD44 was signifcantly less in cells treated with 0.50 μM of cisplatin (p < 0.05), 0.25 mg/ mL of STE, and 0.50 μM of cisplatin combined (p < 0.05) and 0.1 mg/mL of RE and 0.50 μM of cisplatin combined (p < 0.001), as well as 0.25 mg/mL of STE, 0.1 mg/mL of RE, and 0.50 μM of cisplatin combined (p < 0.05).Tere were signifcantly fewer cells expressing CD44 in cells treated with 0.1 mg/mL of RE and 0.50 μM of cisplatin combined compared to cells treated with cisplatin alone (p < 0.05).
Te average percentage of cells expressing CD44 following treatment with Taxol and Taxol-extract combinations is shown in Figure 10(c).Compared to untreated cells, there were signifcantly fewer cells expressing CD44 in cells treated with 0.01 μM of Taxol (p < 0.001), 0.25 mg/mL of STE, and 0.01 μM of Taxol combined (p < 0.0001) and 0.1 mg/mL of RE and 0.01 μM of Taxol combined (p < 0.05), as well as 0.25 mg/mL of STE, 0.1 mg/mL of RE, and 0.01 μM of Taxol combined (p < 0.01).Signifcantly fewer cells expressed CD44 when treated with 0.25 mg/mL of STE and 0.01 μM of Taxol combined compared to cells treated with Taxol alone (p < 0.05).

Discussion
In this research paper, we present the results of the anticancer properties of STE and RE, two well-tolerated NHPs, as well as their interaction with standard chemotherapeutics.
We have shown that both STE and RE induce apoptosis in triple-negative breast cancer at low doses; they enhance the anticancer activity of cisplatin and Taxol indicating positive interaction.Furthermore, these extracts with a complex mixture of bioactive compounds target the mitochondrial (STE) and oxidative vulnerability (RE) of cancer cells.Importantly, these extracts are efective in killing cancer stem cells and sensitize cancer stem cells to Taxol or cisplatin.Tese results demonstrate that STE and RE have the potential to be a great nontoxic supplemental therapy along with chemotherapy for triple-negative breast cancer patients and could also have prevention of relapse as they target cancer stem cells.
Induction of apoptosis by STE and RE selectively in cancer cells was clearly demonstrated by Annexin V binding assay and propidium staining in a dose-dependent manner.Purifed compounds from these plant materials e.g., epigallocatechin-3-gallate in tea extract and carnosic acid, and rosmarinic acid in rosemary extract, have been shown to induce apoptosis in diferent cancers [40,42].Our hypothesis is that the total extract may contain multiple compounds that may target multiple pathways to selectively induce apoptosis in cancer cells.Additionally, the total extracts like STE and RE are characterized as natural health products that are already safe for human consumption.Most importantly, we explored the possibility of these extracts to be used in combination with chemotherapeutic drugs.Both STE and RE induced a signifcant percentage of apoptosis at very low doses (0.05-0.1 mg/mL).Other than the AVPI staining, the morphology of treated cells clearly indicated apoptotic features, such as nuclear condensation and blebbing.Interestingly, at similar doses, noncancerous healthy cells, normal colon mucosal cells (NCM-460), did not show any increase in apoptosis following treatment with these extracts, indicating the selective nature of these extracts for cancer cells (Figure 5).
Recently, several studies have found a variety of NHPs to have anticancer activity.However, medical professionals remain hesitant as the interactions between chemotherapeutics and NHPs are mostly uncharacterized.If NHPs should eventually be administered in clinical settings, the extracts must, at the very least, have no inhibitory efects on chemotherapeutics.
Importantly, our fndings show that STE positively interacted with both cisplatin and Taxol against triplenegative breast cancer as when combined with cisplatin or Taxol, the STE-chemo group induced more apoptosis than the chemotherapeutics did on their own.Combining the chemotherapeutics with STE allows for the administration of much lower doses of the drugs while achieving even greater anticancer activity.Terefore, the herb-drug combination therapy mitigates the toxicity associated with high doses of chemotherapeutics.Tese fndings are very important for developing STE as an adjuvant to standard chemotherapy.
Te most exciting results are found in the herb-herb interaction groups.Combining 0.10 mg/mL of STE with 0.025 mg/mL of RE was, by far, the most efective combination therapy.On its own, each extract induced apoptosis in around 30% of breast cancer cells, but the combination resulted killed Evidence-Based Complementary and Alternative Medicine around 75%. Teir positive interaction goes beyond an additive efect.Tese results indicate that a combination of these two herbal extracts may provide an alternative cancer therapy similar to or better than chemotherapeutics.Overall, we demonstrated that these nontoxic herbal extracts can be combined as a supplement to the chemo regimen where they can enhance the anticancer activity of chemotherapy.In addition, combined treatment with just the two herbal extracts has also the potential of being a very efective cancer therapy for TNBC.Te positive interaction of STE and RE with chemo was also demonstrated by wound healing assays that assesses the inhibition of proliferation and migration of cancer cells by anticancer agents [31].While these extracts on their own were able to inhibit proliferation and migration (indicated by an increased gap in the wound compared to the control), cisplatin and Taxol on their own had very little efect.Most interestingly, a combination of the extracts with chemotherapeutics was more efective in increasing the gap width compared to either chemotherapeutic alone.Tese results further confrm the efcacy of the herbal extracts on their own as well as their positive interactions with chemotherapies.
Mechanistically, NHPs may achieve their selective anticancer activity by exploiting the vulnerabilities that are unique to cancerous cells.Notably, the hyperactive metabolic activity present in cancerous cells renders them susceptible to oxidative stress and mitochondrial depolarization.Indeed, some of recent research has shown these vulnerabilities in cancer cells [45].We have demonstrated that RE's anticancer activity is reliant on the induction of ROS in breast cancer cells.We found that 0.05 mg/mL of RE induced signifcant production of ROS in triple-negative breast cancer.Cisplatin, Taxol, and STE did not induce any ROS on their own.However, when combined with RE, the RE-chemo groups induced ROS when compared to the control.
Te important question is whether the induction of oxidative stress is critical for the induction of apoptosis by RE?We found that RE-induced apoptosis is dependent on the production of ROS as shown by the inhibition of REinduced apoptosis by N-acetylcysteine (NAC), an antioxidant.Terefore, rosemary is dependent on the production of ROS to induce apoptosis in triple-negative breast cancer.STE treatment did not induce ROS production, and STEinduced apoptosis was not afected by NAC.Interestingly, the STE-RE group did not result in any ROS production.Te lack of ROS production in the herb-herb group might be indicative of some sort of inhibitory efect between the extracts.However, the analysis of cell viability showed that STE and RE were at the very least enhancing each other, if not synergizing.One possible explanation may be that STE may be displaying protective efects related to ROS.As mentioned in the introduction, the catechins in STE are known to interact with and quench ROS in vitro.
Mitochondrial vulnerability of cancer cells could be also targeted by these extracts.When we assessed the efect of STE and RE on mitochondrial potential in cancer cells, we observed that STE causes mitochondrial dysfunction to induce apoptosis as indicated by the TMRM assay.We found that 0.10 mg/mL of STE completely collapsed the MMP, as there was almost 0% red fuorescence.Every combination group involving STE displayed similar results.Terefore, neither RE nor the chemotherapeutics inhibited or enhanced STE's ability to target the mitochondria.
Rosemary, cisplatin, and Taxol did not afect the mitochondria on their own.However, the RE-cisplatin and RE-Taxol groups depolarized the MMP when compared to the control.Interestingly, none of the individual components of this combination group could target the MMP, but when combined, there were depolarizing efects.Tis suggests that while RE cannot target the mitochondria on its own, it may potentially be sensitizing cells to mitochondrial depolarization.
As usual, fuorescent microscopy was conducted in parallel with quantitative data analysis to serve as additional evidence.In Figure 9(b), each treatment group is accompanied by two side-by-side images.On the left, both the blue and red fuorescent channels are merged together.Meanwhile, the right shows the red fuorescent channel on its own.Again, red fuorescence indicates healthy, mitochondria.Te control group is characterized by signifcant red fuorescence, while the STE groups display minimal to no red fuorescence.In contrast, RE's red fuorescence is comparable with the control.Terefore, STE dissipated the MMP, while RE did not.
Te extracts targeting diferent pathways is a scientifcally interesting fnding as it speaks on their level of selectivity.Even more, being able to target specifc pathways may be useful if a cell line is found to be more resilient against certain therapies.Perhaps the synergistic efects of the STE-RE group arise from two pathways being simultaneously targeted.Further characterization of their anticancer activity will help with the scientifc validation of these extracts.It is therefore important to continue characterizing the mechanisms of action of both STE and RE.
Tus, STE targets the MMP while RE targets the ROS pathway.As hypothesized, the extracts achieve selective anticancer activity by exploiting the vulnerabilities unique to cancer.Inducing ROS levels beyond the baseline that cellular protection mechanisms can cope with may initiate PCD.As explained by the Warburg efect in the introduction, the overreliance of cancerous cells on glycolysis makes their mitochondria susceptible to depolarization.STE and RE may provide a promising way to selectively target triplenegative breast cancer.

Sensitivity of MDA-MB-231 Stem Cells to STE and Extract-Chemotherapeutic
Combinations.Cancer stem cells have been the focus of research as these cells would be responsible for cancer relapse.CD44, a stem cell surface marker, has been shown to be highly expressed in the MDA-MB-231 cell line [23,24,26].We have evaluated the sensitivity of the cancer stem cells in TNBC to STE and RE.Te average percentage of CSCs in control untreated MDA-MB-231 cells reported in our study (29.05% ± 3.92%) is in agreement with values previously reported 18 Evidence-Based Complementary and Alternative Medicine in the literature [46].Compared to untreated cells, cells treated with STE had a signifcantly lower percentage of CSCs after 48 h.RE on its own did not have a signifcant efect on CSCs.Te percentage of CD44-positive cells compared to the control was signifcantly reduced in cells treated with cisplatin alone, cisplatin combined with STE, cisplatin combined with RE, and cisplatin combined with both extracts.Te percentage of CSCs was signifcantly less in cells treated with RE combined with cisplatin compared to cells treated with cisplatin alone (Figure 10(b)).Tese results suggest that RE enhanced the efcacy of cisplatin and achieved greater CSC death than the drug alone.
Tere was no signifcant diference in the percentage of CSCs remaining between cells treated with cisplatin alone and cells treated with cisplatin combined with STE or between cells treated with cisplatin alone and cells treated with a combination of cisplatin and both extracts.Tis suggests that the greatest synergistic efect was observed when adding RE to cisplatin while STE and STE + RE had no synergistic efect.Importantly, this suggests that neither RE, STE, or both extracts combined interfered with the chemotherapeutic treatment from achieving CSC death.
Te percentage of CD44-positive cells was signifcantly lower in cells treated with Taxol, a combination of STE and Taxol, a combination of RE and Taxol, and a combination of both extracts and Taxol than the untreated control.Most importantly, there was a signifcantly lower percentage of CSCs remaining after treatment with STE and Taxol combined than cells treated with Taxol alone.Tis suggests that STE positively interacted with Taxol, achieving signifcantly higher levels of CSC's death than the drug administered alone.All treatments except RE alone signifcantly reduced the percentage of CSCs compared to untreated cells, suggesting that CSCs are sensitive to STE alone as well as to a combination therapy with both extracts and common chemotherapeutics Taxol and cisplatin.Multiple studies report that CSCs can repopulate tumour masses following treatment with chemotherapeutics such as cisplatin and exhibit chemotherapeutic resistance [10,14,15].Previous studies also suggested that diferential survival of CSCs following chemotherapeutic treatment can increase the percentage of CSCs in progeny [14,15].Since these NHPs are well-tolerated and can be administered long-term, our results suggest that STE alone or in combination with RE can be administered post chemotherapeutic treatment to target CSCs in patients, thus reducing the chance of tumour mass repopulation and cancer relapse.Tese fndings have implications for improving TNBC patient's prognosis because the observed synergistic herb-drug efects could allow for lower doses of a chemotherapeutic to be administered.Smaller doses of chemotherapeutics would decrease the onset of adverse side efects such as nausea and vomiting and would lower the risk of secondary infections that risk being lethal [47,48].Tus, a treatment comprised of either herbdrug combination has the potential to be well-tolerated, less toxic, and more efective at killing CSCs than current treatments, making it a strong candidate for long-term use in TNBC patients.Future studies should investigate whether CSCs develop resistance to treatment with RE and cisplatin combined or treatment with STE and Taxol combined.Future studies should also evaluate whether administering an herb-drug cocktail may be efective in avoiding the development of chemotherapeutic resistance, which would have further implications for reducing the risk of tumour relapse and treatment resistance.
In conclusion, both extracts, STE and RE induced apoptosis in MDA-MB-231 cells in a dose-and timedependent manner.STE interacted positively with conventional chemotherapeutics Taxol and cisplatin, while RE did not interact with them at all.Terefore, combining STE with chemotherapeutics will result in greater anticancer efects.As well, this combination will allow for reduced chemotherapeutic dosages, thereby lowering the risk of toxic side efects.Most importantly, the combination of STE and RE displayed synergistic efects in terms of reducing cell viability in breast cancer.Te STE-RE group was just as efective as the STE-RE-chemo group, thus removing the need for the drugs.With regard to their mechanisms of action, we found that STE induced apoptosis by targeting the MMP, while RE acted by inducing the production of ROS.Furthermore, cancer stem cells were also found to be sensitive to treatment with STE and a STE-RE combination.Notably, CSCs were more sensitive to treatment with a REcisplatin combination compared to cisplatin alone, as well as a STE-Taxol combination compared to Taxol alone.Tese potential synergistic efects against CSCs have implications for reducing the rate of tumour relapse and treatment resistance in TNBC patients and should be further explored in future studies.
Our results suggest that STE and RE can provide breast cancer patients with a sustainable and efective therapeutic regimen.Importantly, they may deal with the hurdles associated with chemotherapeutics, mainly, adverse efects negatively impacting a patient's health.Te development of STE and RE as anticancer therapies may serve to improve the quality of life and long-term healthcare outcomes of breast cancer patients.

Figure 1 :
Figure 1: Synthite tea extract induces apoptosis in breast cancer in a dose-dependent and time-dependent manner.MDA-MB-231 cells were treated with various concentrations of STE at (a) 24 hours and (b) 48 hours.Te graphed results were obtained by using image-based cytometry to assess the percentage of cells positive with fuorescence associated with Annexin V (green), PI (red), and both (yellow) or negative for both Annexin V and PI (blue).Values are expressed as a mean ± SD from three independent experiments.STE treatment groups are compared with a negative control of ddH 2 O. Brightfeld images were taken at 200x or 400x magnifcation using ToupLite software with the OMAX A35100U Microscope.Images are representative of the three independent experiments.Statistical calculations were performed by using a two-way ANOVA multiple comparison test.* p < 0.05 vs control, * * p < 0.01 vs control, * * * p < 0.001 vs the control, and * * * * p < 0.0001 vs control.(c) Fluorescent microscopy of STE apoptosis induction in MDA-MB-231 cells at 48 hours.Fluorescent images were stained with Annexin V (green), PI (red), and Hoechst (blue) at 200x magnifcation.Scale bar is 100 microns.Images are representative of three independent experiments.

Figure 2 :
Figure 2: Rosemary extract induces apoptosis in breast cancer in a dose-dependent manner.MDA-MB-231 cells were treated with various concentrations of RE at (a) 24 hours and (b) 48 hours.Te graphed results were obtained by using image-based cytometry to assess the percentage of cells positive with fuorescence associated with Annexin V (green), PI (red), and both (yellow) or negative for both Annexin V and PI (blue).Values are expressed as a mean ± SD from three independent experiments.RE treatment groups are compared with a negative control of DMSO.Brightfeld images were taken at 200x or 400x magnifcation using ToupLite software with the OMAX A35100U microscope.Images are representative of three independent experiments.Statistical calculations were performed by using a two-way ANOVA multiple comparison test.* p < 0.05 vs control, * * p < 0.01 vs control, * * * p < 0.001 vs the control, and * * * * p < 0.0001 vs control.(c) Fluorescent microscopy of RE apoptosis induction in MDA-MB-231 cells at 48 hours.Fluorescent images were stained with Annexin V (green), PI (red), and Hoechst (blue) at 200x magnifcation.Scale bar is 100 microns.Images are representative of three independent experiments.

Figure 4 .
Figure 4. Te extracts positively interacted with each other and with conventional chemotherapeutics.Te statistical signifcance of cell viability was analyzed for every combination therapy and its individual components.Compared to the control, cell viability was signifcantly reduced in cells treated with 0.10 mg/mL of STE (p < 0.0001), 0.025 mg/mL of RE (p < 0.05), and a combination of STE + RE (p < 0.0001).Treatment with 0.10 mg/mL of STE + 0.025 mg/mL of RE signifcantly reduced cell viability compared to treatment with either extract alone (p < 0.00001 for both comparisons).Cell viability was signifcantly reduced compared to the control in cells treated with 0.50 µM of cisplatin (p < 0.01), cisplatin combined with 0.10 mg/mL of STE (p < 0.0001), cisplatin combined with 0.025 mg/mL of RE (p < 0.01), and cisplatin combined with both extracts (p < 0.0001).Compared to cells treated with cisplatin alone, cell viability was signifcantly reduced in cells treated with cisplatin + STE (p < 0.00001) and cisplatin + STE + RE (p < 0.00001).Tere was no signifcant diference in cell viability in cells treated with cisplatin + RE compared to cells treated with cisplatin alone and cells treated with RE alone.Cell viability was signifcantly reduced in cells treated with cisplatin + STE + RE compared to cells treated with cisplatin alone (p < 0.00001), STE alone (p < 0.00001), and RE alone (p < 0.00001), STE + cisplatin (p < 0.00001), and RE + cisplatin (p < 0.00001).Tere was no signifcant diference in cell viability between cells treated with cisplatin + STE + RE and cells treated with STE + RE.Cell viability was signifcantly reduced compared to the control in cells treated with 0.01 µM of Taxol (p < 0.05), Taxol combined with 0.10 mg/mL of STE (p < 0.0001), Taxol combined with 0.025 mg/mL of RE (p < 0.05), and Taxol combined with both extracts (p < 0.0001).Compared to cells treated with Taxol alone, cell viability was signifcantly reduced in cells treated with Taxol + STE (p < 0.00001).Tere was no signifcant diference in cell viability in cells treated with STE + Taxol compared to cells treated with STE alone.Tere was no signifcant diference in cell viability in cells treated with Taxol + RE compared to cells treated with Taxol alone and cells treated with RE alone.Cell viability was signifcantly reduced in cells treated with Taxol + STE + RE compared to cells treated with Taxol alone (p < 0.00001), STE alone (p < 0.00001), and RE alone (p < 0.00001), STE + Taxol (p < 0.01), and RE + Taxol (p < 0.00001).Tere was no signifcant diference in cell viability between cells treated with Taxol + STE + RE and cells treated with STE + RE.
Cells.Te selectivity of both Synthite tea (Figure5(a)) and rosemary (Figure5(b)) extracts was assessed by using the noncancerous cell line, NCM-460.Varying doses of both extracts were individually administered and assessed at 48 hours.Te Annexin V and propidium iodide (AVPI) assay was used to determine whether these NHPs had adverse efects on noncancerous cells.Te cells were stained with the fuorescent apoptotic-marking dyes, Annexin V (AV) and propidium iodide (PI).

Figure 5 :
Figure5: Synthite tea and rosemary extracts are both selective for breast cancer cells.NCM-460 cells were treated with varying concentrations of (a) STE and (b) RE (0.01 mg/mL-0.25 mg/mL) for 48 hours.Results were obtained by using image-based cytometry to assess the percentage of cells positive with fuorescence associated with Annexin V (green), PI (red), and both (yellow) or negative for both Annexin V and Pi (blue).Values are expressed as a mean ± SD from three independent experiments.* p < 0.05 vs control, * * p < 0.01 vs control, and * * * * p < 0.0001 vs control.

3. 7 .
Mitochondrial Depolarization.Cancerous mitochondria serve as attractive targets for therapies that aim to specifcally target cancer.Mitochondrial dysfunction may ultimately lead to the initiation of programmed cell death.Te fuorescent TMRM assay was used to monitor mitochondrial stability and

Figure 7 :Figure 8 :
Figure 7: RE induces ROS production in triple-negative breast cancer.(a) MDA-MB-231 breast cancer cells were treated with H2DCFDA following treatments with Taxol and cisplatin individually and in combination with STE and RE.Results were obtained by using imagebased cytometry to assess the percentage of cells positive with DCF, which indicates the generation of ROS.Hydrogen peroxide served as a positive control.Treatment groups were compared with the negative control DMSO.Values are expressed as a mean ± SD from three independent experiments.Statistical calculations were performed by using a two-way ANOVA multiple comparison test.* p < 0.05 vs control, * * p < 0.01 vs control, and * * * * p < 0.0001 vs control.(b) Fluorescent microscopy of triple-negative breast cancer cells stained with H2DCFDA (green) and counterstained with Hoechst (blue) at 200x magnifcation.Cells fuorescing green indicate the generation of ROS.Images are representative of three independent experiments.

Figure 9 :
Figure 9: STE is dependent on the depolarization of cancerous mitochondrial membrane potential to induce apoptosis.(a) MDA-MB-231 cells were treated with chemotherapeutics Taxol and cisplatin individually and in combination with STE and RE and assessed at 48 hours.Results were obtained by using image-based cytometry to assess the percentage of cells positive with fuorescence associated with mitochondrial membrane potential (TMRM, fuoresces red).Treatment groups were compared with the negative control DMSO.Values are expressed as a mean ± SD from three independent experiments.* p < 0.05 vs control, * * p < 0.01 vs control, and * * * * p < 0.0001 vs control.(b) Fluorescent microscopy of triple-negative breast cancer cells stained with TMRM (red) and counterstained with Hoechst (blue) at 200x magnifcation.Overlays of both fuorescent channels are on the left, and the red channel alone is on the right.Images are representative of three independent experiments.

Figure 10 :
Figure 10: Immunofuorescence staining of stem cells with CD44 stem cell surface markers in cells treated with STE, RE, cisplatin, Taxol, and herb-drug combinations at 48 hours.Bars represent the average percentage of cells expressing CD44 for each group (n � 3) in MDA-MB-231 at hours following treatment.Error bars represent the positive and negative SD of each group.* p < 0.05, * * p < 0.01, * * * p < 0.001, and * * * * p < 0.0001 compared to the control.‡ p < 0.05 compared to cisplatin alone.† p < 0.05 compared to Taxol alone.Micrographs are overlays of fuorescence and brightfeld images at 200x magnifcation.Scale bar is 100 μm.Each micrograph is representative of the three independent experiments.Cells were stained with Hoechst 33342 (blue) and CD44 (green).(a) Immunofuorescence staining of CSCs with CD44 in cells treated with STE, RE, and a combination of STE and RE.(b) Immunofuorescence staining of stem cells with CD44 stem cell surface markers in cells treated with cisplatin and herb-cisplatin combinations.(c) Immunofuorescence staining of stem cells with CD44 stem cell surface markers in cells treated with Taxol and herb-Taxol combinations.

4
[31]ence-Based Complementary and Alternative Medicine 2.8.Wound Healing Assay.MDA-MB-231 cells were grown to 80% confuency in 6-well tissue culture plates.Cells were scratched with a sterile P200 pipette tip to create a cell-free gap.Loosely attached cells were removed by PBS washing.Cells were treated with 0.1 mg/mL of STE, 0.50 μM of cisplatin, 0.01 μM of Taxol, and both herb-drug combinations.Cells in the negative control received no treatment.Te progression of migration was photographed at 96 hours under fuorescence and bright-feld microscopy following staining with Hoechst 33342 (Molecular Probes, Eugene, OR, USA).Te wound healing closure distance by the migrated cells was measured and averaged over four independent experiments for each treatment group.Te protocol used is similar to that of previously published work[31].
2.9.Immunofuorescence Staining for Cancer Stem Cells.Cells were treated with 0.50 μM of cisplatin, 0.25 mg/mL of STE combined with 0.1 mg/mL of RE, and diferent combinations of the extracts with the drugs cisplatin + RE, cisplatin + STE, cisplatin + RE + STE, taxol + RE, taxol + STE, and taxol + STE + RE.Cells were plated in 8-well tissue culture plates and stained with the standard immunofuorescence protocol.In short, cells were washed with 1x PBS and fxed with 3.7% formaldehyde for 15 minutes.Cells were then washed with 1x PBS and permeabilized with 0.15% Triton X-100 (Sigma-Aldrich, USA) for 2 minutes.Cells were washed with tris-bufered saline (TBS), blocked with 5% bovine serum albumin (BSA) (BioShop Canada Inc., Canada), incubated on a rocker for one hour, and again washed with TBS.A 1 : 1000 dilution of the primary antibody anti-mouse CD44 (obtained from Abcam, Canada, catalogue number ab6124) was prepared in TBS and added to each well, and cells were incubated on a rocker for another hour.Cells were then washed with TBS and incubated on the rocker for 5 minutes three times.A 1 : 1000 dilution of the secondary antibody Alexa Fluor 488 donkey anti-mouse IgG (H + L) (Life Technologies Corporation, Oregon, catalogue number A10042) was prepared in TBS and added to each well, and cells were incubated on a rocker for another hour.Cells were again washed with TBS and incubated on the rocker for 5 minutes three times.Cells were then stained with Hoechst 33342 (Molecular Probes, Eugene, OR, USA).
(a)) was used to further characterize the efects that RE and STE have on oxidative stress in breast cancer.We observed that