Curcumin Analog, HO-3867, Induces Both Apoptosis and Ferroptosis via Multiple Mechanisms in NSCLC Cells with Wild-Type p53

Over the last decade, researchers have paid more and more attention to the natural compound curcumin for its potential application in anticancer therapy. However, the application of curcumin has been limited owing to its rapid metabolism in the body. HO-3867, a stable curcumin analog, shows potent antitumor activities against various tumor cells. Yet, information on HO-3867's impact on non-small-cell lung cancer (NSCLC) cells is lacking. Herein, we evaluated the cytotoxicity of HO-3867 in NSCLC cells. We discovered that HO-3867 suppressed the viability of NSCLC cells containing wild-type p53. In NSCLC cells, HO-3867 promotes both apoptosis and ferroptosis, the latter of which is a newly discovered mode of cell death. Mechanically, HO-3867-induced apoptosis relied on the inhibition of Mcl-1 and Bcl-2 and the upregulation of Bax. Moreover, NSCLC cells undergo ferroptosis when treated with HO-3867 via activating the p53-DMT1 axis and suppressing GPX4. Additionally, HO-3867 caused an accumulation of reactive oxygen species (ROS) in NSCLC in a way that was dependent on the presence of iron. Our findings point to the possibility that HO-3867 might be employed as a therapeutic agent for treating NSCLC.


Introduction
Around 2 million new cases of lung cancer are diagnosed each year, with 1.8 million deaths globally due to the disease [1]. Histopathologically, non-small-cell lung cancer (NSCLC) accounts for the vast majority of lung cancer diagnoses (representing 80-85% of all cases) [2]. Up to this point, the primary therapeutic choices for NSCLC have been surgery, radiation, chemotherapy, and immunotherapy [3]. Te overall prognosis of NSCLC individuals is still unfavorable, despite the evolving advances that have been achieved in the treatment of NSCLC. As a consequence, innovative efective therapeutic techniques are urgently required.
Programmed cell death (PCD) alludes to the death of cells in an organized manner that is meticulously modulated by a wide range of endogenous genes. PCD is implicated in a variety of physiological activities such as tumorigenesis.
Te most studied form of PCD is apoptosis mainly hallmarked by chromatin condensation, cellular shrinkage, DNA fragmentation, formation of apoptotic body, and membrane blebbing [4]. Te apoptotic process is controlled by numerous proteins, such as those belonging to the Bcl-2 family [5]. Unlike apoptosis, necroptosis, and autophagy, the recently discovered ferroptosis is a distinctive PCD mechanism [6]. Ferroptosis is hallmarked by the formation of lipid peroxides in a process dependent on the presence of iron, which subsequently promotes the accumulation of reactive oxygen species (ROS) in cells and, ultimately, results in the death of cells as a result of oxidative stress [7].
Mounting data indicate that defective ferroptosis is implicated in carcinogenesis [8]. Terefore, triggering ferroptosis is a viable technique for eliminating cancer cells, particularly those that are insensitive to apoptosis [9].
In past decades, the strong potential of natural products to destroy cancer cells while exhibiting relatively low toxicity has garnered considerable interest in cancer therapy. Curcumin, the active ingredient of Curcuma longa L, has been found to exhibit anticancer actions against multiple cancers, one of them being lung cancer [10]. Although curcumin is safe even at large dosages in humans, its rapid metabolism in the body has limited its clinical use [11]. To improve the outcomes, various adjuvants and structural analogs of curcumin have been developed. Among them, the new diarylidenyl piperidone (DAP)-inspired analog HO-3867 promotes growth arrest and apoptosis in a myriad of different tumor cell lines, even those from breast, colon, liver, lung, and ovarian cancers, without evident toxicity to normal cells [12,13].
Nevertheless, research on HO-3867's impact on NSCLC cells has not been extensively conducted so far. Terefore, in this research, we systematically examined the mechanisms behind the antitumor efects of HO-3867 on NSCLC cells. Sigma-Aldrich supplied all other routine chemicals.

Cell
Culture. Te American Type of Culture Collection (ATCC, USA) supplied the human NSCLC cell lines (H460, PC-9, H1975, A549, and H1299). A549 p53 KO cells (cat no: ab276092) was ordered from Abcam. H460 p53 KO cells were a generous gift from Dr. Qingfeng Li (Fudan University, China). Te STR test was used to verify the authenticity of the cell lines, and mycoplasma testing was performed routinely. Te culture medium comprised RPMI1640 with 10% fetal bovine serum (FBS, Gibco, USA) and 100 U/ml penicillin/streptomycin (Sigma-Aldrich). Te cells were maintained in an environment containing 5% CO 2 at a temperature of 37°C.

Cell Viability
Assay. Te MTT test was employed to ascertain the cell viability, as earlier reported [14]. For 12 h at 37°C, the cells were inoculated onto 96-well plates at a density of 3 × 10 4 cells/mL, with fve parallel wells in each group. After that, the cells were subjected to HO-3867 treatment in a variety of concentrations and timeframes (24 h, 48 h, and 72 h). Next, following the addition of 10 μl of MTT to each well, they were allowed to incubate at 37°C for additional two hours. Te fndings were presented in the form of a ratio to the absorbance value recorded at 570 nm for the cells serving as controls. Tere were three separate repetitions for the experiment.

Cell Death Measurement.
Te Annexin V-FITC Apoptosis detection kit (BD Bioscience, USA) was adopted to measure cell death following the directions stipulated by the manufacturer. Succinctly, cells were harvested at various time points following treatment followed by the resuspension of precipitate in 400 μl 1 × binding bufer. After that, 5 μl Annexin V-FITC/PI was introduced into the cells, and then, they were allowed to incubate for 20 min at room temperature (RT) in the darkness. For each assessment, 1 × 10 4 cells were counted and examined via fow cytometry (BD Biosciences, USA) to determine cell death.
2.6. Caspase-3 Activities Assay. Caspase-3 Activity Assay Kit (cat no: ab252897, Abcam) was employed to determine the relative activities of caspase-3 activity, as instructed by the manufacturer.

Quantitative RT-PCR.
Trough the use of the TRIzol reagent (Beijing Solarbio Science and Technology Co., Ltd.) as directed by the manufacturer, total RNA was isolated from the cells. Te levels of messenger RNA (mRNA) produced by distinct genes were measured with the assistance of a frst-strand cDNA synthesis kit (cat no: K1611) and SYBR Green Master Mix kit (cat no: A25741, Applied Biosystems; Termo Fisher Scientifc, Inc.) in conformity with the instructions stipulated by the manufacturer. Te ABI 7500 Fast real-time PCR system (Applied Biosystems; Termo Fisher Scientifc, Inc.) was employed for the qRT-PCR procedure. For thermocycling, we applied the following procedures and parameters: initially, the protein was denatured at 95°C for 10 minutes, and then, it was subjected to 35 cycles for 15 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C, and a fnal extension for 5 mins at 72°C. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was measured and it served as an internal control. Evidence-Based Complementary and Alternative Medicine

Iron Level
Assessment. An iron assay kit (ab83366, Abcam) was utilized to determine relative Fe 2+ levels in compliance with the specifcations provided by the manufacturer. Succinctly, overnight cultures were started from cells (1 × 10 5 cells/well) sown in 6-well plates. After that, diferent treatments were applied to the cells for 24 hours. Cells were homogenized with 100 μL iron assay bufer and centrifuged at a rate of 13,000 × g for 10 min at 4°C, and the pellet was then discarded. To prevent the transformation of Fe 3+ to Fe 2+ , the iron reducer included in the kit was introduced into the supernatant. Te components were stirred carefully and left to incubate for 30 minutes in the darkness on ice. After adding 100 μL of Fe 2+ probe, samples were allowed to incubate for 1 hour at RT, away from light. A microplate reader (BioTek, USA) was employed to measure the absorbance at 593 nm. Tere were three separate repetitions for the experiment.

Assessment of ROS.
A ROS detection assay kit (ab287839, Abcam) was employed to determine ROS levels in compliance with the instructions from the manufacturer. In brief, cells (1 × 10 5 cells/well) were cultured throughout the night using 6-well plates. After that, the cells were then subjected to various treatments for 24 h. For 30 minutes at 37°C, cells were exposed to 10 μM DCFH-DA in FBS-free media before being rinsed thrice. Te ROS level was determined by fow cytometry (BD Biosciences, USA) with cell counts of 1 × 10 4 cells for each measurement.

Measurement of Glutathione (GSH).
A GSH assay kit (cat no: ab239727, Abcam) was employed to determine the relative level of GSH as per the package recommendation. In brief, 1 × 10 5 cells/well were seeded into 6-well plates and allowed to incubate in the darkness for 12 hours. Diferent treatments were applied to the cells for 24 hours. After homogenizing the cells in a solution containing 50 mM MES and 1 mM EDTA, the cells were centrifuged at a rate of 13,000 x g for 10 minutes at 4°C followed by the removal of the pellet. After mixing the supernatant with the GSH detection solution and incubating for 25 minutes at RT, the results were read. After transferring the reaction solution to a 96-well plate, the microplate reader (BioTek, USA) was used to read the fndings at a wavelength of 412 nm. Tere were three separate repetitions for the experiment.
2.11. Western Blots. Te RIPA lysis bufer (Beyotime, China) was utilized in the preparation of total cell lysates. Te BCA Protein Assay Kit (Beyotime, China) was applied to determine the concentrations of protein as directed by the manufacturer. Te proteins were extracted using an SDS-PAGE gel at a concentration of 10%, and then, they were transferred onto a PVDF membrane (BD Biosciences). After blocking the membrane using nonfat milk at a concentration of 5%, it was allowed to incubate with primary antibodies for a full night at 4°C. After being rinsed in PBS thrice, the membrane was allowed to incubate in an incubation solution containing the necessary secondary antibody for one hour at RT. Te enhanced chemiluminescence (ECL) system (Merck Millipore, Darmstadt, Germany) was utilized to facilitate the visualization of the fndings.
2.12. Statistical Analysis. Te software SPSS 25.0 (IBM, USA) was employed to execute all analyses of statistical data. Te data were reported using mean ± SD. We made a comparison between the two groups with the unpaired two-tailed Student's t-test, and comparisons among multiple subgroups were carried out with a one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test. A P value of <0.05 (two-tailed) indicated the signifcance level.

HO-3867 Suppressed the Viability of NSCLC Cells with
Wild-Type p53. values confrmed that H1299, A549 p53 KO, and H460 p53 KO cells are less sensitive to HO-3867 than NSCLC cell lines with wild-type p53 ( Figure 1(i)). Collectively, these fndings illustrated that the cytotoxic action of HO-3867 against NSCLC cells relies on p53.

NSCLC Cells Undergo Caspase-Dependent Apoptosis
When Treated with HO-3867. Cell death in NSCLC was studied to assess whether it may be triggered by HO-3867. Varying concentrations of HO-3867 (10 μM, 20 μM, and 40 μM) were employed to treat H460 and A549 cells for 24 h. In addition, Annexin V-FITC staining was employed to measure cell death. Cell death in NSCLC was dosagedependently promoted by HO-3867 ( Figure 2(a)). Several cell death inhibitors (z.VAD.FMK, Nec-1, Fer-1, and Lip-1) were employed to confrm the specifc mode of cell death produced by HO-3867. As depicted in Figure 2 Collectively, these fndings suggest that HO-3867 induces both apoptosis and ferroptosis in NSCLC cells.

Overexpression of Mcl-1 and Bcl-2 or Downregulation of Bax Reduced HO-3867-Induced Apoptosis in NSCLC Cells.
After

HO-3867 Induces Accumulation of Iron in NSCLC Cells.
Ferroptosis inhibitors were shown to reduce HO-3867-induced cell death; therefore, we assessed the involvement of HO-3867 in ferroptosis induction in NSCLC cells. We found that HO-3867 dosage-dependently induced iron accumulation in NSCLC cells (Figure 4(a)). Multiple iron chelators (DFO, Dp44mT, and dexrazoxane HCl) were used to verify iron's involvement in HO-3867-triggered cell death. As depicted in Figure 4

Upregulation of DMT1 in a p53-Dependent Way Is Critical for HO-3867-Mediated Ferroptosis in NSCLC Cells.
Several diferent DMT1 inhibitors (salinomycin, ebselen, and S-benzylisothiourea) were used to determine whether or not DMT1 was implicated in the HO-3867-triggered cell death. As illustrated in Figure 5(a), DMT1 inhibitors successfully abrogated the upregulation of HO-3867-induced iron in NSCLC cells. DMT1 inhibitors also remarkably suppressed HO-3867-mediated cell apoptosis in NSCLC cells ( Figure 5(b)). Two siRNAs against DMT1 were used to silence DMT1 in NSCLC cells, allowing for additional investigation into DMT1's function in HO-3867-triggered cell death ( Figure 5(c)). Te downregulation of DMT1 also reduced the HO-3867-induced increase in iron levels in NSCLC cells ( Figure 5(d)). DMT1 silencing also remarkably suppressed HO-3867-mediated apoptosis in NSCLC cells ( Figure 5(e)). As we noticed that p53-defcient cells are insensitive to HO-3867, we assessed the status of p53 in NSCLC cells. Results illustrated that HO-3867 PFTα also reduced iron levels in NSCLC cells after exposure to HO-3867 ( Figure 5(h)). Moreover, PFTα remarkably suppressed HO-3867-mediated death in NSCLC cells ( Figure 5(i)). Tereafter, we assessed the status of DMT1 in p53 KO cells after exposure to HO-3867. Figure 5(j) demonstrates that treatment with HO-3867 attenuated the upregulation of DMT1 in p53 KO cells. In addition, the upregulation of iron levels was also attenuated in p53 KO cells ( Figure 5(k)). HO-5637-mediated cell death was also much less in p53 KO cells than in NSCLC cells with wildtype p53 ( Figure 5(l)). Taken together, these fndings indicate that HO-3867 upregulates DMT1 in NSCLC cells in a p53-dependent way.

HO-3867 Iron-Dependently Induced Accumulation of ROS in NSCLC Cells.
Te generation of ROS is another critical event in the process of ferroptosis [15]. In particular, we examined the diference in ROS levels in NSCLC cells both before and after they were exposed to HO-3867. As illustrated in Figure 6(a), a dosagedependent elevation in ROS levels was observed in NSCLC cells when following the HO-3867 treatment. ROS scavenger (NAC) was capable of abrogating HO-3867's ability to produce an increase in ROS production in NSCLC cells (Figure 6(a)). NAC also remarkably suppressed HO-3867-mediated death of NSCLC cells (Figure 6(b)). Noteworthy, it was also observed that DFO attenuated HO-3867-triggered ROS upregulation in NSCLC cells (Figure 6(a)). To evaluate if HO-3867-induced upregulation of ROS is iron-dependent, more iron chelators were examined. Te iron chelators Dp44mT and dexrazoxane HCl also reduced HO-3867-induced upregulation of ROS in NSCLC cells (Figure 6(c)). After that, we assessed the involvement of DMT1 in the HO-3867-triggered increase in ROS production in NSCLC cells. As can be seen in Figure 6(d), the levels of ROS produced by NSCLC cells decreased remarkably when DMT1 inhibitors were used. Similarly, the enhancement of ROS generation that was caused by HO-3867 in NSCLC cells was also decreased by DMT1 silencing (Figure 6(e)). Collectively, as evidenced by these data, it appears that HO-3867 causes an accumulation of ROS in NSCLC cells in a way that is reliant on iron.

HO-3867 Treatment Reduced GPX4 Expression in NSCLC Cells.
Te depletion of GSH is another essential hallmark of ferroptosis [15]. Tus, we examined the GSH level in NSCLC cells that had been pretreated with HO-3867. As can be seen in Figure 7(a), HO-3867 caused a dosage-dependent decrease in the level of GSH that was generated by NSCLC cells. SLC7A11 is a multipass transmembrane protein that regulates the transportation of cysteine, a rate-limiting precursor for the production of GSH [16]. Glutathione peroxidase 4 (GPX4) belongs to the GPX family of enzymes. It utilizes reduced GSH as a cofactor in its detoxifcation of lipid peroxide, thereby, inhibiting ferroptosis [17]. Tus, we evaluated how HO-3867 afected the concentrations of GPX4 and SLC7A11 in NSCLC cells. As per the outcomes of RT-PCR, HO-3867 contributed to a dosage-dependent decrease in the level of GPX4 mRNA found in NSCLC cells (Figure 7(b)). Even thorough HO-3867 did not have an impact on the levels of SLC7A11 mRNA in NSCLC cells (Figure 7(c)). Similar results were obtained using Western blotting, which showed that HO-3867 dosagedependently decreased the protein levels of GPX4 in NSCLC cells, although it had no impact on the protein levels of SLC7A11 (Figure 7(d)). Overexpressing GPX4 in NSCLC cells allowed us to investigate whether or not it plays a role in the ferroptosis that is triggered by HO-3867 (Figure 7(e)). After being exposed to HO-3867, our fndings showed that GPX4 overexpression increased GSH levels in NSCLC cells (Figure 7(f )). Furthermore,  (f ) A549 and H460 cells were incubated with indicated doses of HO-3867 for 24 h, and protein levels of p53 were assayed by Western blotting. (g) A549 and H460 cells were incubated with HO-3867 (40 μM) in combination with PFTα (10 μM) or not for 24 h, and protein levels of DMT1 were assayed by Western blotting. (h) A549 and H460 cells were treated as above, and iron levels were assayed. (i) Cellular death was assayed. (j) A549 and H460 wild-type (WT) or p53 KO (p53-/-) cells were treated with or without HO-3867 (40 μM) for 24 h, and the indicated proteins were assayed by Western blotting. (k) Cells were treated as above, and iron levels were assayed. (l) Cellular death was assayed. Data are presented as mean ± SD. * P < 0.01 and * * P < 0.01.

Discussion
Cell death is remarkably involved in many diferent physiological processes, including the progression of cancers and their subsequent treatments [5]. In this research, NSCLC cells were exposed to an analog of curcumin called HO-3867, which was then tested for its antitumor roles. We discovered that the use of HO-3867 decreased the viability of NSCLC cells with wild-type p53. Additionally, the HO-3867 treatment causes NSCLC cells to undergo both ferroptosis and apoptosis.
Extensive research has focused on apoptosis, a kind of cell death marked by caspase activation [18]. We established that HO-3867 induces apoptosis in NSCLC cells. At frst, NSCLC cell death caused by HO-3867 was blocked by the pan-caspase inhibitor z.VAD.FMK. Second, caspase-3 activities assays showed that HO-3867 promoted the activities of caspase-3. Tird, Western blotting revealed that HO-3867 induced caspase-3 cleavage. Tese results are consistent with past research demonstrating that HO-3867 triggers apoptosis in several cancer cells, such as those of the pancreas, ovaries, oral cavity, and osteosarcoma cells [19][20][21][22]. Apoptosis may occur through two diferent pathways, the intrinsic and extrinsic/mitochondrial pathways. Te Bcl-2 family of proteins performs a fundamental function in modulating the intrinsic pathway [23]. We showed that the antiapoptotic Bcl-2 members Mcl-1 and Bcl-2 were  (e) A549 and H460 cells were transfected as indicated for 24 h, and protein levels of GPX4 were assayed. (f ) A549 and H460 cells were transfected as indicated for 24 h, cells were treated with or without HO-3867 (40 μM) for another 24 h, and GSH levels were assayed. (g) A549 and H460 cells were transfected as indicated for 24 h, then cells were treated with or without HO-3867 (40 μM) for another 24 h, and cellular death was assayed. Data are presented as mean ± SD. * P < 0.01 and * * P < 0.01. downregulated in NSCLC cells after treatment with HO-3867. Tese data demonstrate that HO-3867 triggers apoptosis via the intrinsic mechanism, which is similar to earlier research that revealed that HO-3867 reduced Bcl-2 and Mcl-1 in ovarian cancer cells [24,25]. Interestingly, another study found that HO-3867 upregulated Bcl-2 in cardiomyocytes [26]. Tis variation might be attributed to diferent cell types. Tus, understanding how HO-3867 regulates Bcl-2 proteins requires additional research.
Emerging research suggests that ferroptosis, a newly discovered mode of cell death, performs an instrumental function in modulating the advancement of many cancers, NSCLC included [27]. Ferroptosis is highly prevalent in lung cancer tissues because of increased levels of ROS and lipid peroxide [28]. Hence, to eradicate NSCLC cells, inhibiting ferroptosis could be an efective method. To date, extensive research has demonstrated that curcumin causes ferroptosis in various cancerous cells such as NSCLC and breast cancer cells [29,30]. Here, we revealed, for the frst time, that HO-3867 could also induce ferroptosis in NSCLC cells. Investigation of the mechanisms underlying HO-3867-induced ferroptosis revealed that HO-3867 activates the p53/ DMT1 axis. Interestingly, HO-3867 has been illustrated to trigger wild-typep53-like efects by covalently binding to mutant p53 [31]. Tis suggests that HO-3867 may exert its efect, at least partially, via the regulation of p53. Tis may also explain why HO-3867 shows little cytotoxicity against p53-defcient cells. We found that HO-3867 caused the upregulation of p53 in NSCLC cells; however, another recent study showed that HO-3867 suppressed p53 levels in ovarian cancer cells [32]. Tis discrepancy reveals the complex effects of HO-3867 on the regulation of p53 and must be clarifed.
Even though systemic iron dysregulation is prevalent in cancer patients, the link between iron and lung cancer risk remains controversial. For instance, several research reports have found that high dietary iron intake remarkably elevates the chance of developing lung cancer [33][34][35]. High iron intake has also been linked to a reduced chance of developing lung cancer [36]. Moreover, iron transportation-related proteins such as TFR, FTL, and FTH were found to be upregulated in lung cancer [37]. We observed that HO-3867 did not change the TFR, FTL, or FTH expression but did enhance the expression of DMT1. DMT1 works as a proton-coupled pump and uses the cell membrane potential for the active transport of iron [38]. Most cells ubiquitously express DMT1, with special prominence in the apical membrane of duodenal enterocytes [39]. Our fndings suggest that DMT1 expression is subject to p53 regulation as the upregulation of DMT1 was abrogated in p53 defciency cells. Tese fndings were consistent with existing literature showing that p53 elevated DMT1 levels in kidney cell lines [40]. In another study, researchers observed that DMT1 expression in hepatocellular carcinoma cells was unafected by p53 [41]. Hence, more investigations are necessary to further elucidate the link between p53 and DMT1. Moreover, we also discovered that DMT1 is essential for the buildup of ROS mediated by HO-3867. Tis is consistent with past research showing that inhibiting DMT1 in pancreatic β cells decreases ROS production and cell death [42]. Our fndings shed new light on DMT1's function in NSCLC therapy.
Another vital fnding of our study is that HO-3867 treatment reduced GPX4 levels in NSCLC cells. Tese data are in line with previous research that found curcuminsuppressed GPX4 levels in NSCLC and breast cancer cells [29,30]. Remarkably, another research report found that ALZ003, another curcumin analog, also reduced GPX4 expression in glioblastoma cells [43]. Although we found that HO-3867 did not afect the expression of SLC7A11, curcumin is known to modulate the levels of SLC7A11 in lung cancer and lung epithelial cells [29,44]. More research studies are warranted to clarify this disparity.
Nonetheless, there are a few drawbacks to this research. First, we did not investigate the anti-NSCLC efects of HO-3867 in vivo owing to constrained time and funding. Hence, the antitumor properties of HO-3867 should be investigated in xenograft models. Second, we found that Mcl-1 and Bcl-2 were both downregulated in response to HO-3867, but the mechanisms behind this phenomenon remain unclear. Also, we did not investigate the mechanisms underlying HO-3867-induced suppression of GPX4. HO-3867 is a STAT3 inhibitor [19]. Many studies suggest that STAT3 participates in the upregulation of GPX4 in various cells [45,46]. Terefore, we hypothesized that HO-3867 downregulated GPX4 via inhibition of STAT3 and did not experimentally confrm it.

Conclusions
Our fndings demonstrate that HO-3867, a curcumin analog, triggers both ferroptosis and apoptosis in p53 wildtype NSCLC cells. Mechanically, HO-3867 induces apoptosis by downregulating Bcl-2 and Mcl-1 levels and upregulating Bax. Also, HO-3867 activates the p53/DMT1 axis to increase iron accumulation, which further causes the accumulation of ROS in NSCLC cells. Moreover, HO-3867 also represses GPX4 in NSCLC cells. Collectively, these results imply that HO-3867 has the potential as a treatment target for NSCLC.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.