The Effect of G. applanatum Crude Polysaccharide Extract on Proinflammatory Cytokines and Proapoptotic Caspases in HeLa Cell Line: An In Vitro Study

Polysaccharide extracts exhibit promise as potential anticancer agents. Among the fungi rich in polysaccharide content, G. applanatum stands out; however, its anticancer activity necessitates further investigation. This study aims to explore the impact of G. applanatum crude polysaccharide (GACP) extract by assessing its effects on cell viability, levels of proinflammatory cytokines such as TNF-α, IFN-γ, IL-2, and IL-12, and levels of proapoptotic markers including caspase-3 and caspase-9, as well as the percentages of necrosis and apoptosis in the HeLa cell line. Employing the HeLa cell line as a research model, four groups were studied: KN (media and DMSO), K+ (doxorubicin 10 μg/mL), P1 (G. applanatum extract 200 μg/mL), and P2 (G. applanatum extract 400 μg/mL). The G. applanatum extract was obtained via boiling distilled water. Anticancer activity was evaluated through the MTT test (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) conducted over three treatment durations (24, 48, and 72 hours). Cytokine levels and caspase-3 and caspase-9 levels were assessed using the ELISA test. Cell apoptosis was determined using the Annexin V-PI biomarker and analyzed through flow cytometry. The MTT test exhibited optimal results at the 48-hour treatment mark. Cytokine level analysis revealed significant reductions in TNF-α, IFN-γ, IL-2, and IL-12 levels (p < 0.005). Concurrently, caspase-3 and caspase-9 levels exhibited substantial increases (p < 0.005). Flow cytometry highlighted the highest percentage of apoptosis in HeLa cells. In conclusion, G. applanatum's polysaccharide extract demonstrates potential as an anticancer and therapeutic agent for cancer treatment.


G. applanatum Crude Polysaccharide Extract Preparation.
G. applanatum specimens were sourced from Tulungagung, East Java, Indonesia.Te extraction procedure, as detailed by Susilo et al. [14], was pursued.Dr. Ni'matuzzahroh conducted the identifcation based on a designated key book [19].In essence, the basidiocarp of G. applanatum was sectioned and air-dried, after which the dried segments were milled into a fne powder.Te powdered material was weighed and subjected to boiling at temperatures between 90 and 100 °C for a duration of 6 hours.Following the boiling phase, the fltrate extracted from G. applanatum underwent centrifugation at 4300 rpm for 5 minutes.Te resulting supernatant was precipitated using absolute ethanol in a 1 : 3 ratio.Tis precipitation process was reiterated three times, and the ensuing pellets were isolated.Te pellets were subsequently dissolved in distilled water and again subjected to centrifugation, following the same approach.Te resultant pellets were freeze-dried to yield the GACP extract.

FTIR Analysis.
For FTIR analysis, the GACP extract was incorporated into potassium bromide (KBr) pellets and assessed using the Nicolet ™ iS20 FTIR Spectrometer.

HeLa Cell
Culture.HeLa cells were procured from Stem Cell Research and Development at Universitas Airlangga.Te cells were cultivated in DMEM and incubated at a temperature of 37 °C in an atmosphere containing 5% CO 2 .Te administration of GACP extract was undertaken based on specifc concentrations allocated to each group, facilitated by a DMSO solvent at levels below 1%.Te treatment doses applied in this investigation encompassed the K− group (negative control: media + HeLa cell line), K+ group (positive control: HeLa cell line + 10 µg/mL doxorubicin), P1 group (HeLa cell line + GACP 200 µg/mL), and P2 group (HeLa cell line + GACP 400 µg/mL).Cells were subsequently cultured for durations of 24, 48, and 72 hours before being harvested to facilitate further assessments.

Cell Viability.
Te evaluation of HeLa cell viability entailed the utilization of the MTT technique (3-(4,5dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) [34].Cells procured from the CO 2 incubator underwent meticulous examination to confrm their uncontaminated status.For cell harvest, cultures achieving an 80% confuence were selected.Postharvest, cell enumeration, and subsequent dilution with complete culture medium occurred.Te cells were then dispensed into a 96-well plate, allotting 5 × 10 3 cells to each well, followed by overnight incubation.Varied concentrations of GACP extract (200 µg/mL and 400 µg/mL) were introduced, employing DMSO as a cosolvent.Tis assembly was then incubated in a 37 °C, 5% CO 2 incubator for a duration of 24 hours.At the conclusion of incubation, 100 μL of MTT reagent (0.5 mg/mL) dissolved in DMEM was appended to each well.A subsequent 3-hour incubation at 37 °C ensued, resulting in the formation of formazan.Te cellular examination was conducted employing an inverted microscope.Subsequent to the distinct appearance of formazan, the introduction of a 10% SDS halt solution in 0.1 N HCl took place.Te plate, enveloped in aluminum foil, was relegated to an obscure setting overnight.Te optical density (OD) values were gauged via a microplate reader at a wavelength of 595 nm.
2.5.Cytokine Levels Analysis.ELISA kits (Bioassay Technology, Shanghai, China) facilitated the quantifcation of cytokine levels in accordance with the manufacturer's stipulations.Each well of a well plate received a total of 40 L of HeLa cell supernatant.Sequentially, 10 μL and 50 μL of 2 Advances in Pharmacological and Pharmaceutical Sciences streptavidin-HRP antibodies were incorporated.A 60minute incubation at 37 °C followed, succeeded by fve washes with washing bufer.Subsequently, 50 μL of substrate solutions A and B were individually introduced to each well.An additional 10-minute incubation at 37 °C under dark conditions ensued.Termination involved the addition of 50 μL of stop solution to each well.Tis treatment was executed across six replications.Within a span of less than 10 minutes, the optical density (OD) values were recorded through an ELISA reader (Termo Scientifc ™ Multiskan ™ GO Microplate Spectrophotometer) at a wavelength of 450 nm.
2.6.Caspase Levels Analysis.Caspase-3 and caspase-9 levels were assessed through the utilization of an ELISA kit (Bioassay Technology, Shanghai, China), adhering to the provided manufacturing protocol.Each well plate was allocated a total of 40 L of supernatant originating from HeLa cell cultures.Subsequent to this, mouse antibody caspase-3 was introduced for the quantifcation of caspase-3 levels, while mouse antibody caspase-9 was employed for the evaluation of caspase-9 levels.A sequential addition of 50 L of streptavidin-HRP followed, culminating in an 1-hour incubation at room temperature.Following this, a thricerepeated washing step was undertaken using a washing bufer.Successively, 50 L of substrate A solution was dispensed into each well plate.Subsequently, substrate B was introduced and incubated in the dark at 37 °C for a duration of 10 minutes.Te conclusion of this process involved the addition of 50 L of stop solution.Tis treatment was executed across six replications.Measurement occurred via an ELISA reader (Termo Scientifc ™ Multiskan ™ GO Microplate Spectrophotometer) at a wavelength of 450 nm.
2.7.Apoptosis Index.Te detection of apoptosis was executed through fow cytometry [35].In 6-well plates, a total of 1 × 10 5 HeLa cells were subjected to incubation with G. applanatum extract and medium under 5% CO 2 at 37 °C overnight.Subsequent to this incubation, the mixture of G. applanatum extract and medium was withdrawn.Te addition of a solution composed of PBS, EDTA, and trypsin transpired in each well, followed by a 2-minute incubation within the CO 2 incubator.Te aspirates from each well were then combined in Eppendorf tubes and centrifuged at 2500 rpm for 5 minutes.Postcentrifugation, cells within each Eppendorf were subjected to a cold PBS wash, followed by centrifugation at 1500 rpm at 4 °C.A suspension of 400 L binding bufer incorporating 5 L annexin V-FITC and 10 L PI was prepared, and a 10-minute incubation at 4 °C in darkness ensued.Tis treatment was executed across six replications.Tis protocol was replicated six times.Flow cytometry analysis was undertaken using guava

Statistical Analysis.
All data were presented as mean ± SD and subsequently subjected to analysis using GraphPad Prism software version 8 (San Diego, CA, USA).
Te one-way ANOVA test was employed to ascertain the signifcance of GACP extract impact.Following this, the Tukey test was administered to highlight signifcant disparities between groups.A statistical signifcance threshold was established at p < 0.05.

FTIR Analysis.
Te FTIR analysis results are depicted in Figure 1.Te analysis unveiled the existence of multiple functional groups through the observation of a total of 41 peaks.Notably, a robust peak at 1631.Importantly, in the positive group and GACP-treated groups, parameters such as TNF-α levels, IFN-c levels, IL-2 levels, and IL-12 levels were signifcantly (p < 0.05) lower than those in the negative control group.Te administration of GACP extract efectively mitigated the increase in TNF-α levels, IFN-c levels, IL-2 levels, and IL-12 levels when compared to the negative control group.Tese results indicated that the GACP extract efectively played a protective role against cancer.Comparative analysis between the efects of doxorubicin and GACP extract on proinfammatory cytokine parameters demonstrated that doxorubicin had a lower impact than GACP extract on HeLa cells.Nevertheless, GACP extract exhibited signifcant effects compared to the negative control group, afrming its potential as an anticancer agent.

Efect of G. applanatum Extract on Caspase-9 and
Caspase-3.Figure 5 illustrates the impact of G. applanatum extract on caspase-9 and caspase-3 levels.Regarding caspase-3 levels, values were found to be 3.60 ± 0.  4 Advances in Pharmacological and Pharmaceutical Sciences caspase-9, the levels were 1.73 ± 0.22 µg/mL for K−, 5.67 ± 1.62 µg/mL for K+, 4.05 ± 0.34 µg/mL for P1, and 4.105 ± 0.37 µg/mL for P2.Tese ELISA assay results strongly indicate that GACP signifcantly increased caspase-9 and caspase-3 levels, implying that GACP potentially induced apoptosis through the classical nuclear DNA damage pathways.Notably, the expression of caspase-9 and caspase-3 in the GACP-treated groups and the positive control group was signifcantly (p < 0.05) higher than that in the negative control group.When comparing the efects of doxorubicin and GACP extract on caspase levels, substantial diferences were observed.Doxorubicin led to higher increases in caspase-9 and caspase-3 levels than those in the GACP-treated groups.Nevertheless, the administration of GACP extract signifcantly increased caspase-9 and caspase-3 levels, underscoring its role in apoptosis induction and afrming its potential as an anticancer agent.

Discussion
Te utilization of natural sources as a safe approach for drug development, especially in cancer treatment, has gained prominence.In this study, GACP was administered to HeLa cells to assess cell viability.Te results of GACP exposure over 24, 48, and 72 hours exhibited varying percentages of cell viability.Notably, the 72-hour exposure yielded higher cell viability percentages compared to 24 and 48 hours at higher doses.Tis condition could be attributed to cell adaptation in HeLa cells.Extended exposure to GACP, such as at 72 hours, may have facilitated cell survival and replication.Te fndings suggest that GACP does not exert toxic efects on HeLa cells.Chronic conditions often lead to increased levels of proinfammatory cytokines.In the case of HeLa cells, they produce proinfammatory cytokines such as TNF-α, IL-6, IL-2, IL-12, and IFN-c to sustain tumor  Data are presented as mean ± SD (n � 6).* * * * p < 0.0001 compared to the negative group (K−).* * * p < 0.001 compared to the negative group (K−).* p < 0.05 compared to the negative group (K−).# p < 0.05 compared to the positive group (K+).K−: negative control; K+: positive control; P1: G. applanatum crude polysaccharide extract 100 µg/mL; P2: G. applanatum crude polysaccharide extract 200 µg/mL.6 Advances in Pharmacological and Pharmaceutical Sciences development and proliferation over the long term [36,37].Moreover, proinfammatory cytokines can drive angiogenesis and metastasis within the tumor microenvironment [21,38].Te activation of NF-κB is a critical factor in promoting proinfammatory cytokine abundance.Te prevention of NF-κB activation has become a signifcant focus in the feld of cancer treatment.In this study, GACP demonstrated the ability to decrease levels of TNF-α, IL-6, IL-2, IL-12, and IFN-c.Te polysaccharides present in GACP are thought to inhibit IKβ phosphorylation, thereby hindering the translocation of NF-κβ.Tis mechanism ultimately results in lower proinfammatory cytokine production by HeLa cells [22,39].Te study also explored the impact of GACP on the apoptosis pathway in HeLa cells.Te decrease in proinfammatory cytokine levels contributed to a reduced survival rate of HeLa cells, thereby initiating the apoptosis process [40][41][42].Tis study aims to examine GACP efects in apoptosis, measured by fow cytometry.Te use of Annexin V-FITC allowed for the detection of early apoptosis, as it can identify phospholipid phosphatidylserine (PS) exposed on the outer plasma membrane.In addition, propidium iodide (PI) was employed to stain nuclear DNA, enabling the identifcation of late apoptosis and necrosis following the loss of cell membrane integrity.Te dose of 400 µg/mL of GACP exhibited the highest percentage of late apoptosis at 48 hours.However, the optimal dose was found to be 200 µg/mL at 24 hours, as it led to 39.2% viable cells without inducing cell toxicity while still inducing apoptosis in 45.6% of HeLa cells.Notably, the percentage of early apoptosis induced by doxorubicin was higher than that induced by GACP treatment or the control group.Tis suggests that GACP holds potential as an anticancer agent, particularly in triggering late apoptosis.Furthermore, the study elucidated the underlying mechanism of GACP-induced apoptosis, particularly through the intrinsic pathway.Te treatment with GACP led to elevated levels of caspase-9 and caspase-3 compared to the control group.Caspase-9, known as an initiator caspase, plays a pivotal role in activating executor caspases, such as caspase-3 [43][44][45].Apoptosis is initiated by an increase in proapoptotic proteins like BAX, BAK1, BIM, BID, and BBC3, which stimulate mitochondria to release cytochrome c into the cytoplasm.Tis triggers the formation of the apoptosome and activation of caspase-3 [25].Te apoptosome forms when cytochrome c binds to Apaf-1, followed by the subsequent joining of procaspase-9 to the apoptosome, leading to its cleavage and activation.Te activation of caspase-9 further stimulates the activation of caspase-3 [26], which orchestrates apoptosis by cleaving and activating proteins crucial for the proteolytic degradation of cancer cells.Ultimately, this process leads to the elimination of cancer cells and the suppression of tumor growth.Te enhancement of caspase-9 and caspase-3 production suggests that GACP holds potential as an anticancer agent, particularly for the treatment of cervical cancer.
Previous studies have underscored the potential therapeutic efects of G. applanatum crude extract in cervical cancer treatment.Tese efects include inducing apoptosis via the intrinsic pathway, inhibiting DNA survival and transcription factor proliferation, causing DNA fragmentation, and curbing the growth and metastatic potential of cervical cancer cells [46].Te existing literature on G. applanatum's anticancer properties further solidifes its potential in cancer therapy, including its toxicity against tumor cells compared to normal cells [47].

Conclusion
In conclusion, the polysaccharide compounds obtained from G. applanatum have the capability to induce apoptosis in HeLa cells by upregulating the levels of caspase-3 and caspase-9.However, to gain a deeper understanding of the underlying molecular mechanisms driven by G. applanatum extract, further investigations are warranted.Further research should prioritize the analysis of protein interactions, protein signaling, and regulatory factors implicated in apoptotic responses, particularly through animal studies (in vivo).and RD supervised the project.All authors contribute to the manuscript and approved the fnal version of the manuscript.