HDAC8-Selective Inhibition by PCI-34051 Enhances the Anticancer Effects of ACY-241 in Ovarian Cancer Cells

HDAC6 is overexpressed in ovarian cancer and is known to be correlated with tumorigenesis. Accordingly, ACY-241, a selective HDAC6 inhibitor, is currently under clinical trial and has been tested in combination with various drugs. HDAC8, another member of the HDAC family, has recently gained attention as a novel target for cancer therapy. Here, we evaluated the synergistic anticancer effects of PCI-34051 and ACY-241 in ovarian cancer. Among various ovarian cancer cells, PCI-34051 effectively suppresses cell proliferation in wild-type p53 ovarian cancer cells compared with mutant p53 ovarian cancer cells. In ovarian cancer cells harboring wild-type p53, PCI-34051 in combination with ACY-241 synergistically represses cell proliferation, enhances apoptosis, and suppresses cell migration. The expression of pro-apoptotic proteins is synergistically upregulated, whereas the expressions of anti-apoptotic proteins and metastasis-associated proteins are significantly downregulated in combination treatment. Furthermore, the level of acetyl-p53 at K381 is synergistically upregulated upon combination treatment. Overall, co-inhibition of HDAC6 and HDAC8 through selective inhibitors synergistically suppresses cancer cell proliferation and metastasis in p53 wild-type ovarian cancer cells. These results suggest a novel approach to treating ovarian cancer patients and the therapeutic potential in developing HDAC6/8 dual inhibitors.     

Trichodermin Induces G0/G1 Cell Cycle Arrest by Inhibiting c-Myc in Ovarian Cancer Cells and Tumor Xenograft-Bearing Mice

Ovarian cancer is a fatal gynecological cancer because of a lack of early diagnosis, which often relapses as chemoresistant. Trichodermin, a trichothecene first isolated from Trichoderma viride, is an inhibitor of eukaryotic protein synthesis. However, whether trichodermin is able to suppress ovarian cancer or not was unclear. In this study, trichodermin (0.5 µM or greater) significantly decreased the proliferation of two ovarian cancer cell lines A2780/CP70 and OVCAR-3. Normal ovarian IOSE 346 cells were much less susceptible to trichodermin than the cancer cell lines. Trichodermin predominantly inhibited ovarian cancer cells by inducing G0/G1 cell cycle arrest rather than apoptosis. Trichodermin decreased the expression of cyclin D1, CDK4, CDK2, retinoblastoma protein, Cdc25A, and c-Myc but showed little effect on the expression of p21^Waf1/Cip1, p27^Kip1, or p16^Ink4a. c-Myc was a key target of trichodermin. Trichodermin regulated the expression of Cdc25A and its downstream proteins via c-Myc. Overexpression of c-Myc attenuated trichodermin’s anti-ovarian cancer activity. In addition, trichodermin decelerated tumor growth in BALB/c nude mice, proving its effectiveness in vivo. These findings suggested that trichodermin has the potential to contribute to the treatment of ovarian cancer.     

A Regulatory Loop Involving miR-200c and NF-κB Modulates Mortalin Expression and Increases Cisplatin Sensitivity in an Ovarian Cancer Cell Line Model

Ovarian cancer is currently the most lethal gynecological cancer. At present, primary debulking surgery combined with platinum-based chemotherapy is the standard treatment strategy for ovarian cancer. Although cisplatin-based chemotherapy has greatly improved the prognosis of patients, the subsequent primary or acquired drug resistance of cancer cells has become an obstacle to a favorable prognosis. Mortalin is a chaperone that plays an important role in multiple cellular and biological processes. Our previous studies have found that mortalin is associated with the proliferation and migration of ovarian cancer cells and their resistance to cisplatin-based chemotherapy. In this study, microRNA (miR)-200b/c downregulated mortalin expression and inhibited the proliferation and migration of the paired cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) epithelial ovarian cancer cell lines. Moreover, miR-200c increased the sensitivity of ovarian cancer cells to cisplatin treatment by regulating mortalin levels. Nuclear factor (NF)-κB directly regulated mortalin and miR-200b/c expression levels, while NF-κB and miR-200b/c jointly regulated the expression of mortalin. The combination of cisplatin and miR-200c significantly enhanced the therapeutic effects on ovarian cancer in vivo, suggesting that miR-200c may serve as a potential therapeutic agent for ovarian cancer.     

Catalpol Suppresses Proliferation and Facilitates Apoptosis of OVCAR-3 Ovarian Cancer Cells through Upregulating MicroRNA-200 and Downregulating MMP-2 Expression

Catalpol is expected to possess diverse pharmacological actions including anti-cancer, anti-inflammatory and hypoglycemic properties. Matrix metalloproteinase-2 (MMP-2) is closely related to the pathogenesis of ovarian cancer. In addition, microRNA-200 (miR-200) can modulate phenotype, proliferation, infiltration and transfer of various tumors. Here, OVCAR-3 cells were employed to investigate whether the effect of catalpol (25, 50 and 100 μg/mL) promoted apoptosis of ovarian cancer cells and to explore the potential mechanisms. Our results demonstrate that catalpol could remarkably reduce the proliferation and accelerate the apoptosis of OVCAR-3 cells. Interestingly, our findings show that catalpol treatment significantly decreased the MMP-2 protein level and increased the miR-200 expression level in OVCAR-3 cells. Further, microRNA-200 was shown to regulate the protein expression of MMP-2 in OVCAR-3 cells. It is concluded that catalpol suppressed cellular proliferation and accelerated apoptosis in OVCAR-3 ovarian cancer cells via promoting microRNA-200 expression levels and restraining MMP-2 signaling.     

Sinomenine Hydrochloride Promotes TSHR-Dependent Redifferentiation in Papillary Thyroid Cancer

Radioactive iodine (RAI) plays an important role in the diagnosis and treatment of papillary thyroid cancer (PTC). The curative effects of RAI therapy are not only related to radiosensitivity but also closely related to the accumulation of radionuclides in the lesion in PTC. Sinomenine hydrochloride (SH) can suppress tumor growth and increase radiosensitivity in several tumor cells, including PTC. The aim of this research was to investigate the therapeutic potential of SH on PTC cell redifferentiation. In this study, we treated BCPAP and TPC-1 cells with SH and tested the expression of thyroid differentiation-related genes. RAI uptake caused by SH-pretreatment was also evaluated. The results indicate that 4 mM SH significantly inhibited proliferation and increased the expression of the thyroid iodine-handling gene compared with the control group (p < 0.005), including the sodium/iodide symporter (NIS). Furthermore, SH also upregulated the membrane localization of NIS and RAI uptake. We further verified that upregulation of NIS was associated with the activation of the thyroid-stimulating hormone receptor (TSHR)/cyclic adenosine monophosphate (cAMP) signaling pathway. In conclusion, SH can inhibit proliferation, induce apoptosis, promote redifferentiation, and then increase the efficacy of RAI therapy in PTC cells. Thus, our results suggest that SH could be useful as an adjuvant therapy in combination with RAI therapy in PTC.     

Antitumor Effects of Selenium

Functions of selenium are diverse as antioxidant, anti-inflammation, increased immunity, reduced cancer incidence, blocking tumor invasion and metastasis, and further clinical application as treatment with radiation and chemotherapy. These functions of selenium are mostly related to oxidation and reduction mechanisms of selenium metabolites. Hydrogen selenide from selenite, and methylselenol (MSeH) from Se-methylselenocyteine (MSeC) and methylseleninicacid (MSeA) are the most reactive metabolites produced reactive oxygen species (ROS); furthermore, these metabolites may involve in oxidizing sulfhydryl groups, including glutathione. Selenite also reacted with glutathione and produces hydrogen selenide via selenodiglutathione (SeDG), which induces cytotoxicity as cell apoptosis, ROS production, DNA damage, and adenosine-methionine methylation in the cellular nucleus. However, a more pronounced effect was shown in the subsequent treatment of sodium selenite with chemotherapy and radiation therapy. High doses of sodium selenite were effective to increase radiation therapy and chemotherapy, and further to reduce radiation side effects and drug resistance. In our study, advanced cancer patients can tolerate until 5000 μg of sodium selenite in combination with radiation and chemotherapy since the half-life of sodium selenite may be relatively short, and, further, selenium may accumulates more in cancer cells than that of normal cells, which may be toxic to the cancer cells. Further clinical studies of high amount sodium selenite are required to treat advanced cancer patients.     

JI017, a Complex Herbal Medication,Induces Apoptosis via the Nox4–PERK–CHOP Axis in Ovarian Cancer Cells

Many anti-cancer drugs, including paclitaxel and etoposide, have originated and been developed from natural products, and traditional herbal medicines have fewer adverse effects and lesser toxicity than anti-tumor reagents. Therefore, we developed a novel complex herbal medicine, JI017, which mediates endoplasmic reticulum (ER) stress and apoptosis through the Nox4–PERK–CHOP signaling pathway in ovarian cancer cells. JI017 treatment increases the expression of GRP78, ATF4, and CHOP and the phosphorylation of PERK and eIF2α via the upregulation of Nox4. Furthermore, it increases the release of intracellular reactive oxygen species (ROS), the production of intracellular Ca²⁺, and the activation of exosomal GRP78 and cell lysate GRP78. Combination treatment using the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG) and JI017 reportedly induces increased ER stress and cell death in comparison to the control; however, knockdown experiments of PERK and CHOP indicated suppressed apoptosis and ER stress in JI017-treated ovarian cancer cells. Furthermore, targeting Nox4 using specific siRNA and pharmacological ROS inhibitors, including N-acetylcystein and diphenylene iodonium, blocked apoptosis and ER stress in JI017-treated ovarian cancer cells. In the radioresistant ovarian cancer model, when compared to JI017 alone, JI017 co-treatment with radiation induced greater cell death and resulted in overcoming radioresistance by inhibiting epithelial–mesenchymal-transition-related phenomena such as the reduction of E-cadherin and the increase of N-cadherin, vimentin, Slug, and Snail. These findings suggest that JI017 is a powerful anti-cancer drug for ovarian cancer treatment and that its combination treatment with radiation may be a novel therapeutic strategy for radioresistant ovarian cancer.     

Metformin and Dichloroacetate Suppress Proliferation of Liver Cancer Cells by Inhibiting mTOR Complex 1

The Warburg effect is important for cancer cell proliferation. This phenomenon can be flexible by interaction between glycolysis and mitochondrial oxidation for energy production. We aimed to investigate the anticancer effects of the pyruvate dehydrogenase kinase inhibitor, dichloroacetate (DCA) and the mitochondrial respiratory complex I inhibitor metformin in liver cancer cells. The anticancer effect of DCA and/or metformin on HepG2, PLC/PRF5 human liver cancer cell lines, MH-134 murine hepatoma cell lines, and primary normal hepatocytes using MTT assay. Inhibition of lactate/ATP production and intracellular reactive oxygen species generation by DCA and metformin was investigated. Inhibition of PI3K/Akt/mTOR complex I was evaluated to see whether it occurred through AMPK signaling. Anticancer effects of a combination treatment of DCA and metformin were evaluated in HCC murine model. The results showed that metformin and DCA effectively induced apoptosis in liver cancer cells. A combination treatment of metformin and DCA did not affect viability of primary normal hepatocytes. Metformin upregulated glycolysis in liver cancer cells, thereby increasing sensitivity to the DCA treatment. Metformin and DCA inhibited mTOR complex I signaling through upregulated AMPK-independent REDD1. In addition, metformin and DCA increased reactive oxygen species levels in liver cancer cells, which induced apoptosis. A combination treatment of metformin and DCA significantly suppressed the tumor growth of liver cancer cells using in vivo xenograft model. Taken together, the combined treatment of metformin and DCA suppressed the growth of liver cancer cells. This strategy may be effective for patients with advanced liver cancer.     

Amplification Target ADRM1: Role as an Oncogene and Therapeutic Target for Ovarian Cancer

Approximately 25,000 ovarian cancers are diagnosed in the U.S. annually, and 75% are in the advanced stage and largely incurable. There is critical need for early detection tools and novel treatments. Proteasomal ubiquitin receptor ADRM1 is a protein that is encoded by the ADRM1 gene. Recently, we showed that among 20q13-amplified genes in ovarian cancer, ADRM1 overexpression was the most highly correlated with amplification and was significantly upregulated with respect to stage, recurrence, and metastasis. Its overexpression correlated significantly with shorter time to recurrence and overall survival. Array-CGH and microarray expression of ovarian cancer cell lines provided evidence consistent with primary tumor data that ADRM1 is a 20q13 amplification target. Herein, we confirm the ADRM1 amplicon in a second ovarian cancer cohort and define a minimally amplified region of 262 KB encompassing seven genes. Additionally, using RNAi knock-down of ADRM1 in naturally amplified cell line OAW42 and overexpression of ADRM1 via transfection in ES2, we show that (1) ADRM1 overexpression increases proliferation, migration, and growth in soft agar, and (2) knock-down of ADRM1 results in apoptosis. Proteomic analysis of cells with ADRM1 knock-down reveals dysregulation of proteins including CDK-activating kinase assembly factor MAT1. Taken together, the results indicate that amplified ADRM1 is involved in cell proliferation, migration and survival in ovarian cancer cells, supporting a role as an oncogene and novel therapeutic target for ovarian cancer.     

A Novel Prostate-Specific Membrane-Antigen (PSMA) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation via Inducing Intrinsic Apoptotic Pathway

Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG’s effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa.     

Metabolomic Identification of Anticancer Metabolites of Australian Propolis and Proteomic Elucidation of Its Synergistic Mechanisms with Doxorubicin in the MCF7 Cells

The combination of natural products with standard chemotherapeutic agents offers a promising strategy to enhance the efficacy or reduce the side effects of standard chemotherapy. Doxorubicin (DOX), a standard drug for breast cancer, has several disadvantages, including severe side effects and the development of drug resistance. Recently, we reported the potential bioactive markers of Australian propolis extract (AP-1) and their broad spectrum of pharmacological activities. In the present study, we explored the synergistic interactions between AP-1 and DOX in the MCF7 breast adenocarcinoma cells using different synergy quantitation models. Biochemometric and metabolomics-driven analysis was performed to identify the potential anticancer metabolites in AP-1. The molecular mechanisms of synergy were studied by analysing the apoptotic profile via flow cytometry, apoptotic proteome array and measuring the oxidative status of the MCF7 cells treated with the most synergistic combination. Furthermore, label-free quantification proteomics analysis was performed to decipher the underlying synergistic mechanisms. Five prenylated stilbenes were identified as the key metabolites in the most active AP-1 fraction. Strong synergy was observed when AP-1 was combined with DOX in the ratio of 100:0.29 (w/w) as validated by different synergy quantitation models implemented. AP-1 significantly enhanced the inhibitory effect of DOX against MCF7 cell proliferation in a dose-dependent manner with significant inhibition of the reactive oxygen species (p < 0.0001) compared to DOX alone. AP-1 enabled the reversal of DOX-mediated necrosis to programmed cell death, which may be advantageous to decline DOX-related side effects. AP-1 also significantly enhanced the apoptotic effect of DOX after 24 h of treatment with significant upregulation of catalase, HTRA2/Omi, FADD together with DR5 and DR4 TRAIL-mediated apoptosis (p < 0.05), contributing to the antiproliferative activity of AP-1. Significant upregulation of pro-apoptotic p27, PON2 and catalase with downregulated anti-apoptotic XIAP, HSP60 and HIF-1α, and increased antioxidant proteins (catalase and PON2) may be associated with the improved apoptosis and oxidative status of the synergistic combination-treated MCF7 cells compared to the mono treatments. Shotgun proteomics identified 21 significantly dysregulated proteins in the synergistic combination-treated cells versus the mono treatments. These proteins were involved in the TP53/ATM-regulated non-homologous end-joining pathway and double-strand breaks repairs, recruiting the overexpressed BRCA1 and suppressed RIF1 encoded proteins. The overexpression of UPF2 was noticed in the synergistic combination treatment, which could assist in overcoming doxorubicin resistance-associated long non-coding RNA and metastasis of the MCF7 cells. In conclusion, we identified the significant synergy and highlighted the key molecular pathways in the interaction between AP-1 and DOX in the MCF7 cells together with the AP-1 anticancer metabolites. Further in vivo and clinical studies are warranted on this synergistic combination.     

Beclin 1 Expression in Ovarian Tissues and Its Effects on Ovarian Cancer Prognosis

Beclin 1 is an autophagy-associated protein involved in apoptosis and drug resistance, as well as various malignancies. We investigated the expression of Beclin 1 protein in ovarian epithelial tissues and correlated it with the prognosis of ovarian cancer. Beclin 1 protein expression was determined using immunohistochemistry in 148 patients with ovarian epithelial cancer, 26 with ovarian borderline tumor, 25 with benign ovarian tumor, and 30 with normal ovarian tissue. The relationships between Beclin 1 protein expression and ovarian cancer pathological characteristics were analyzed. The risk factors for ovarian cancer prognosis were analyzed using Cox’s regression model. A survival curve was plotted from the follow-up data of 93 patients with ovarian cancer to analyze the effects of Beclin 1 expression on the prognosis of ovarian cancer. The positive rates of Beclin 1 were significantly higher in ovarian epithelial cancer (148) and borderline tumor (26) than in benign ovarian tumor (25) or normal ovarian tissue (30) (all p < 0.001). The surgical stage and Beclin 1 expression were both independent risk factors for ovarian cancer prognosis (both p < 0.05). Patients with high Beclin 1 levels showed better survival than those with low Beclin 1 levels (p = 0.009). Beclin 1 protein is upregulated in ovarian epithelial cancer and is a prognostic factor of ovarian cancer.     

Evodiamine Induces Apoptosis and Enhances TRAIL-Induced Apoptosis in Human Bladder Cancer Cells through mTOR/S6K1-Mediated Downregulation of Mcl-1

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, has been considered as a new strategy for anti-cancer therapy. In this study, we demonstrated that evodiamine, a quinolone alkaloid isolated from the fruit of Evodia fructus, induced apoptosis and enhanced TRAIL-induced apoptosis in human bladder cancer cells. To elucidate the underlying mechanism, we found that evodiamine significantly reduced the protein levels of Mcl-1 in 253J and T24 bladder cancer cells, and overexpression of this molecule attenuated the apoptosis induced by evodiamine alone, or in combination with TRAIL. Further experiments revealed that evodiamine did not affect the mRNA level, proteasomal degradation and protein stability of Mcl-1. On the other hand, evodiamine inhibited the mTOR/S6K1 pathway, which usually regulates protein translation; moreover, knockdown of S6K1 with small interfering RNA (siRNA) effectively reduced Mcl-1 levels, indicating evodiamine downregulates c-FLIP through inhibition of mTOR/S6K1 pathway. Taken together, our results indicate that evodiamine induces apoptosis and enhances TRAIL-induced apoptosis possibly through mTOR/S6K1-mediated downregulation of Mcl-1; furthermore, these findings provide a rationale for the combined application of evodiamine with TRAIL in the treatment of bladder cancer.     

Anti-Epidermal Growth Factor Receptor (EGFR) Antibodies Overcome Resistance of Ovarian Cancer Cells to Targeted Therapy and Natural Cytotoxicity

The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.     

BET-Inhibitor I-BET762 and PARP-Inhibitor Talazoparib Synergy in Small Cell Lung Cancer Cells

Small cell lung cancer (SCLC) is an aggressive type of lung cancer with high mortality that is caused by frequent relapses and acquired resistance. Despite that several target-based approaches with potential therapeutic impact on SCLC have been identified, numerous targeted drugs have not been successful in providing improvements in cancer patients when used as single agents. A combination of targeted therapies could be a strategy to induce maximum lethal effects on cancer cells. As a starting point in the development of new drug combination strategies for the treatment of SCLC, we performed a mid-throughput screening assay by treating a panel of SCLC cell lines with BETi or AKi in combination with PARPi or EZH2i. We observed drug synergy between I-BET762 and Talazoparib, BETi and PARPi, respectively, in SCLC cells. Combinatorial efficacy was observed in MYCs-amplified and MYCs-wt SCLC cells over SCLC cells with impaired MYC signaling pathway or non-tumor cells. We indicate that drug synergy between I-BET762 and Talazoparib is associated with the attenuation HR-DSBR process and the downregulation of various players of DNA damage response by BET inhibition, such as CHEK2, PTEN, NBN, and FANCC. Our results provide a rationale for the development of new combinatorial strategies for the treatment of SCLC.     

Cellular Regulation of Kynurenic Acid-Induced Cell Apoptosis Pathways in AGS Cells

Kynurenic acid was included in the three compounds (caffeic acid, chlorogenic acid, and kynurenic acid) that showed high antioxidant and anti-inflammatory potential among the phenolic compounds contained in Gynura procumbens. In this study, the mechanism of cancer cell death induced by kynurenic acid (KYNA), which has the highest molecular binding affinity, in the gastric cancer cell line AGS was confirmed in molecular docking analysis. KYNA showed the most cancer cell death effect on AGS cells among several gastric cancer cell lines (MKN, AGS, and SNU). AGS cells were used for later experiments, and KYNA concentrations of 0, 150, 200, and 250 µM were used. KYNA inhibited cell migration and proliferation in AGS cells in a concentration-dependent manner. G2/M phase cell cycle arrest and reduction of related proteins (Cdc25C, CDK1 and CyclinB1) were confirmed in KYNA-treated AGS cells. Apoptosis of KYNA-treated AGS cells was confirmed by Annexin V/propidium iodide (PI) staining flow cytometry analysis. As a result of morphological chromatin condensation through DAPI (4′,6-diamidino-2-phenylindole), intense blue fluorescence was confirmed. The mechanism of apoptosis induction of KYNA-treated AGS cells was confirmed by western blotting. In the extrinsic pathway, apoptosis induction markers FasL, Fas, and Caspase-3 and -8 were increased in a concentration-dependent manner upon KYNA treatment. In the intrinsic pathway, the expression of anti-apoptotic factors PI3K, AKT, and Bcl-xL was down-regulated, and the expression of apoptosis-inducing factors BAD, Bak, Bax, Cytochrom C, and Caspase-9 was up-regulated. Therefore, in the present study, we strongly imply that KYNA induces apoptosis in AGS gastric cancer cells. This suggests that KYNA, a natural compound, could be the basis for drug for the treatment of gastric cancer.     

Effects of Ruxolitinib and Calcitriol Combination Treatment on Various Molecular Subtypes of Breast Cancer

The anticancer effects of ruxolitinib and calcitriol against breast cancer were reported previously. However, the effect of ruxolitinib and calcitriol combination treatment on various molecular subtypes of breast cancer remains unexplored. In this study, we used MCF-7, SKBR3, and MDA-MB-468 cells to investigate the effect of ruxolitinib and calcitriol combination treatment on cell proliferation, apoptosis, cell cycle, and cell signaling markers, in vitro and in vivo. Our results revealed the synergistic anticancer effect of ruxolitinib and calcitriol combination treatment in SKBR3 and MDA-MB-468 cells, but not in MCF-7 cells in vitro, via cell proliferation inhibition, apoptosis induction, cell cycle arrest, and the alteration of cell signaling protein expression, including cell cycle-related (cyclin D1, CDK1, CDK4, p21, and p27), apoptosis-related (c-caspase and c-PARP), and cell proliferation-related (c-Myc, p-p53, and p-JAK2) proteins. Furthermore, in the MDA-MB-468 xenograft mouse model, we demonstrated the synergistic antitumor effect of ruxolitinib and calcitriol combination treatment, including the alteration of c-PARP, cyclin D1, and c-Myc expression, without significant drug toxicity. The combination exhibited a synergistic effect in HER2-enriched and triple-negative breast cancer subtypes. In conclusion, our results suggest different effects of the combination treatment of ruxolitinib and calcitriol depending on the molecular subtype of breast cancer.