Cisplatin Plus Cetuximab Inhibits Cisplatin-Resistant Human Oral Squamous Cell Carcinoma Cell Migration and Proliferation but Does Not Enhance Apoptosis.

Cisplatin is among the most widely used anticancer drugs used in the treatment of several malignancies, including oral cancer. However, cisplatin treatment often promotes chemical resistance, subsequently causing treatment failure. Several studies have shown that epidermal growth factor receptors (EGFRs) play a variety of roles in cancer progression and overcoming cisplatin resistance. Therefore, this study focused on EGFR inhibitors used in novel targeted therapies as a method to overcome this resistance. We herein aimed to determine whether the combined effects of cisplatin and cetuximab could enhance cisplatin sensitivity by inhibiting the epithelial-to-mesenchymal transition (EMT) process in cisplatin-resistant cells. In vitro analyses of three cisplatin-resistant oral squamous cell carcinoma cells, which included cell proliferation assay, combination index calculation, cell cytotoxicity assay, live/dead cell count assay, Western blot assay, propidium iodide staining assay, scratch assay, and qRT-PCR assay were then conducted. Our results showed that a cisplatin/cetuximab combination treatment inhibited cell proliferation, cell motility, and N-cadherin protein expression but induced E-cadherin and claudin-1 protein expression. Although the combination of cisplatin and cetuximab did not induce apoptosis of cisplatin-resistant cells, it may be useful in treating oral cancer patients with cisplatin resistance given that it controls cell motility and EMT-related proteins.

Increased FOXM1 Expression by Cisplatin Inhibits Paclitaxel-Related Apoptosis in Cisplatin-Resistant Human Oral Squamous Cell Carcinoma (OSCC) Cell Lines.

Cisplatin and paclitaxel are commonly used to treat oral cancer, but their use is often limited because of acquired drug resistance. Here, we tested the effects of combined cisplatin and paclitaxel on three parental (YD-8, YD-9, and YD-38) and three cisplatin-resistant (YD-8/CIS, YD-9/CIS, and YD-38/CIS) oral squamous cell carcinoma (OSCC) cell lines using cell proliferation assays and combination index analysis. We detected forkhead box protein M1 (FOXM1) mRNA and protein expression via real-time qPCR and Western blot assays. Cell death of the cisplatin-resistant cell lines in response to these drugs with or without a FOXM1 inhibitor (forkhead domain inhibitory compound 6) was then measured by propidium iodide staining and TdT dUTP nick end labeling (TUNEL) assays. In all six OSCC cell lines, cell growth was more inhibited by paclitaxel alone than combination therapy. Cisplatin-induced overexpression of FOXM1 showed the same trend only in cisplatin-resistant cell lines, indicating that it was associated with inhibition of paclitaxel-related apoptosis. In summary, these results suggest that, in three cisplatin-resistant cell lines, the combination of cisplatin and paclitaxel had an antagonistic effect, likely because cisplatin blocks paclitaxel-induced apoptosis. Cisplatin-induced FOXM1 overexpression may explain the failure of this combination.

The role of p53 status on the synergistic effect of CKD-602 and cisplatin on oral squamous cell carcinoma cell lines.

The purpose of this study was to evaluate the synergistic apoptotic effect of CKD-602 in combination with cisplatin on different p53 statuses of oral squamous cell carcinoma (OSCC) cell lines, YD-8, YD-9, and YD-38. MTT assays were used to evaluate the inhibitory effect of the treatments modality on cell growth. The combination index was calculated using CompuSyn software. Detection of cell death was carried out using the propidium iodide (PI)/RNase staining assay, Annexin V/PI double staining assay, and Western blotting. Combination treatment using CKD-602 and cisplatin inhibited proliferation and increased the apoptotic effect on the three OSCC cell lines. Apoptotic cell death was detected in the cell lines, and significant synergistic effects of CKD-602 in combination with cisplatin were observed despite the differences in p53 status. From these results, it was suggested that the combination of CKD-602 with cisplatin might be a potential therapeutic strategy for OSCC. In particular, cell line-specific therapy is necessary because of the differences in treatment response based on p53 status.

Upregulation of MDR- and EMT-Related Molecules in Cisplatin-Resistant Human Oral Squamous Cell Carcinoma Cell Lines.

Cisplatin is one of the major drugs used in oral cancer treatments, but its usage can be limited by acquired drug resistance. In this study, we established three cisplatin-resistant oral squamous cell carcinoma (OSCC) cell lines and characterized them using cell viability assays, qPCR, Western blotting, FACS, immunofluorescence, and wound healing assays. Three OSCC cell lines (YD-8, YD-9, and YD-38) underwent long-term exposure to cisplatin, eventually acquiring resistance to the drug, which was confirmed by an MTT assay. In these three newly established cell lines (YD-8/CIS, YD-9/CIS, and YD-38/CIS), overexpression of multidrug resistance (MDR)-related genes was detected by qPCR and Western blotting. The cell lines displayed an increase in the functional activities of breast cancer resistance protein (BCRP) and multidrug resistance protein1 (MDR1) by rhodamine 123 and bodipy FL prazosin accumulation assays. Moreover, the cisplatin-resistant cells underwent morphological changes, from round to spindle-shaped, increased expression of epithelial-to-mesenchymal transition (EMT)-related molecules such as N-cadherin, and showed increased cell migration when compared with the parental cell lines. These results suggest that these newly established cell lines have acquired drug resistance and EMT induction.

SH003 enhances paclitaxel chemosensitivity in MCF-7/PAX breast cancer cells through inhibition of MDR1 activity.

Paclitaxel is an anti-cancer drug for treating cancer, but paclitaxel resistance is reported in cancer cells. Multidrug resistance (MDR) is related with the epithelial-to-mesenchymal transition (EMT) mechanism, which plays a key role in cancer metastasis. Moreover, EMT mechanism is connected to tamoxifen resistance in breast cancer cells. Consequently, oncologists are interested in finding new MDR1 inhibitors originating from herbal medicines to have less side-effect. Here, we investigated an inhibition effect of SH003 on MDR1 activity in paclitaxel-resistant MCF-7/PAX breast cancer cells. Our results showed that paclitaxel did not inhibit a proliferation in paclitaxel-resistant MCF-7 breast cancer cells. Paclitaxel-resistant MCF-7 cells showed an increase of MDR1 activity, which was confirmed by measuring an amount of accumulated rhodamine 123 in the cells. Also, qRT-PCR and Western blot assays confirmed that paclitaxel-resistant MCF-7 cells exhibited high MDR1 expression level. Furthermore, paclitaxel-resistant MCF-7 cells showed mesenchymal morphology with alterations of EMT markers, and acquired tamoxifen resistance with a decrease of ERα expression. We also found that a combinatorial treatment of SH003 and paclitaxel in paclitaxel-resistant MCF-7 cells caused apoptosis in synergistic manner, which was due to SH003 inhibition of MDR1 expression. Therefore, SH003 could be a potential agent for overcoming MDR in drug-resistant cancer cells.

Rhus verniciflua Stokes (RVS) and butein induce apoptosis of paclitaxel-resistant SKOV-3/PAX ovarian cancer cells through inhibition of AKT phosphorylation.

BACKGROUND: Rhus verniciflua Stokes (RVS) belongs to the Anacardiaceae family and traditionally used for cancer treatment. RVS and butein, a major compound of RVS, were known to induce apoptosis via AKT inhibition in cancer cells. Thus, in this study, we investigated the effect of RVS and its derivative compounds (fisetin, quercetin, butein) on cell death in SKOV-3/PAX cells. METHODS: The 80 % ethanol extract of RVS and its derivative compounds (fisetin, quercetin, butein) were prepared. The cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Apoptotic cells were detected by staining with propidium iodide (PI) and Annexin V-fluorescein isothiocyanate/7-aminoactinomycin D (Annexin V-FITC/7-AAD). The expression level of intracellular signaling related-proteins in apoptosis and growth were measured by western blot assay. RESULTS: We found that RVS and butein suppressed the growth of SKOV-3/PAX cells in a dose-dependent manner. We also found that RVS and butein produced the cleavage of caspase-9, -8, -3, and PARP. Similarly, sub-G1 phase and Annexin V-FITC positive cells were increased by RVS and butein. Moreover, RVS and butein significantly reduced AKT phosphorylation in SKOV-3/PAX cells. PI3K inhibitor LY294002 caused PARP cleavage supporting our finding. CONCLUSION: Our data clearly indicate that RVS and butein induce apoptosis of SKOV-3/PAX cells through inhibition of AKT activation. RVS and butein could be useful compounds for the treatment for paclitaxel resistant-ovarian cancer.

Decursin in Angelica gigas Nakai (AGN) Enhances Doxorubicin Chemosensitivity in NCI/ADR-RES Ovarian Cancer Cells via Inhibition of P-glycoprotein Expression.

Angelica gigas Nakai (AGN, Korean Dang-gui) is traditionally used for the treatment of various diseases including cancer. Here, we investigated multidrug-resistant phenotype-reversal activities of AGN and its compounds (decursin, ferulic acid, and nodakenin) in doxorubicin-resistant NCI/ADR-RES ovarian cancer cells. Our results showed that a combination of doxorubicin with either AGN or decursin inhibited a proliferation of NCI/ADR-RES cells. These combinations increased the number of cells at sub-G1 phase when cells were stained with Annexin V-fluorescein isothiocyanate. We also found that these combinations activated caspase-9, caspase-8, and caspase-3 and increased cleaved PARP level. Moreover, an inhibition of P-glycoprotein expression by either AGN or decursin resulted in a reduction of its activity in NCI/ADR-RES cells. Therefore, our data demonstrate that decursin in AGN inhibits doxorubicin-resistant ovarian cancer cell proliferation and induces apoptosis in the presence of doxorubicin via blocking P-glycoprotein expression. Therefore, AGN would be a potentially novel treatment option for multidrug-resistant tumors by sensitizing to anticancer agents. Copyright © 2016 John Wiley & Sons, Ltd.

Ethanol extract of paeonia suffruticosa Andrews (PSE) induced AGS human gastric cancer cell apoptosis via fas-dependent apoptosis and MDM2-p53 pathways.

BACKGROUND: The root bark of Paeonia suffruticosa Andrews (PSE), also known as Moutan Cortex, has been widely used in Asia to treat various diseases. The molecular mechanisms by which PSE exerts its anti-oxidant and anti-inflammatory activities are well known, but its anti-cancer activity is not yet well understood. Here, we present evidence demonstrating that PSE can be used as a potent anti-cancer agent to treat gastric cancer. METHODS: The effects of the ethanol extract of PSE on cell proliferation were determined using an MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) assay. Cell cytotoxicity induced by the PSE extact is measured using an LDH leakage assay. Flow cytometry was used to analyze the cell cycle and to measure the subG0/G1 apoptotic cell fraction. Apoptosis induced by the PSE extact is also examined using a DNA fragmentation assay. Western blot analysis is used to measure the levels of apoptotic proteins such as Fas receptor, caspase-8, caspase-3, PARP, Bax, Bcl-2, MDM2, and p53. RESULTS: This study demonstrated that treating AGS cells with the PSE extact significantly inhibited cell proliferation and induced cytotoxicity in a dose- and time-dependent manner. The PSE extract also induced apoptosis in AGS cells, as measured by flow cytometry and a DNA fragmentation assay. We found that the PSE extract induced apoptosis via the extrinsic Fas-mediated apoptosis pathway, which was concurrent with the activation of caspases, including caspase-8 and caspase-3, and cleavage of PARP. The MDM2-p53 pathway also played a role in the apoptosis of AGS cells that was induced by the PSE extract. CONCLUSIONS: These results clearly demonstrate that the PSE extact displays growth-suppressive activity and induces apoptosis in AGS cells. Our data suggest that the PSE extact might be a potential anti-cancer agent for gastric cancer.

Induction of Fas-mediated extrinsic apoptosis, p21WAF1-related G2/M cell cycle arrest and ROS generation by costunolide in estrogen receptor-negative breast cancer cells, MDA-MB-231.

Costunolide (C(15)H(20)O(2)) is a sesquiterpene lactone that was isolated from many herbal medicines and it has diverse effects according to previous reports. However, the anti-cancer effects and the mechanism of actions are still unknown in breast cancer. In this study, we first observed that costunolide inhibits cell growth in a dose-and time-dependent manner. To examine the mechanism by which costunolide inhibits cell growth, we checked the effect of costunolide on apoptosis and the cell cycle. Costunolide induced apoptosis through the extrinsic pathway, including the activation of Fas, caspase-8, caspase-3, and degradation of PARP. However, did not have the same effect on the intrinsic pathway as revealed by analysis of mitochondrial membrane potential (Δψm) with JC-1 dye and expression of Bcl2 and Bax proteins level. Furthermore, costunolide induced cell cycle arrest in the G2/M phase via decrease in Cdc2, cyclin B1 and increase in p21WAF1 expression, independent of p53 pathway in p53-mutant MDA-MB-231 cells and increases Cdc2-p21WAF1 binding. In addition, costunolide had a slight induced effect on ROS generation. Among the mechanisms of p21WAF1 induction examined, costunolide-induced increase in p21WAF1 expression was related with protein stability and ROS generation. Through this study we confirm that costunolide induces G2/M cell cycle arrest and apoptotic cell death via extrinsic pathway in MDA-MB-231 cells suggesting that it could be a promising anticancer drug especially for ER-negative breast cancer.

Assessing Gene Expression Related to Cisplatin Resistance in Human Oral Squamous Cell Carcinoma Cell Lines.

Cisplatin-based chemotherapy has been effectively used to treat oral cancer, but treatment often fails owing to the development of drug resistance. However, the important gene expression alterations associated with these resistances remain unclear. In this study, we aimed to identify the gene expressions related to cisplatin resistance in oral squamous cell carcinoma (OSCC) cell lines. RNA samples were obtained from three cisplatin-resistant (YD-8/CIS, YD-9/CIS, and YD-38/CIS) and -sensitive (YD-8, YD-9, and YD-38) cell lines. Global gene expression was analyzed using RNA sequencing (RNA-Seq). Differentially expressed genes were determined. Based on the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, functional enrichment and signaling pathways analyses were performed. Candidate genes selected from RNA-Seq analysis were validated by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The YD-8/CIS and YD-9/CIS samples had very similar expression patterns. qRT-PCR analysis was performed on selected genes commonly expressed between the two samples. The expression levels of 11 genes were changed in cisplatin-resistant samples compared with their parental samples; several of these genes were related to cell adhesion molecules and proteoglycans in cancer pathways. Our data provide candidate genes associated with cisplatin resistance in OSCC, but further study is required to determine which genes have an important role. Nevertheless, these results may provide new ideas to improve the clinical therapeutic outcomes of OSCC.

Apigenin overcomes drug resistance by blocking the signal transducer and activator of transcription 3 signaling in breast cancer cells.

Drug resistance in chemotherapy is a serious obstacle for the successful treatment of cancer. Drug resistance is caused by various factors, including the overexpression of P­glycoprotein (P­gp, MDR1). The development of new, useful compounds that overcome drug resistance is urgent. Apigenin, a dietary flavonoid, has been reported as an anticancer drug in vivo and in vitro. In the present study, we investigated whether apigenin is able to reverse drug resistance using adriamycin­resistant breast cancer cells (MCF­7/ADR). In our experiments, apigenin significantly decreased cell growth and colony formation in MCF­7/ADR cells and parental MCF­7 cells. This growth inhibition was related to the accumulation of cells in the sub­G0/G1 apoptotic population and an increase in the number of apoptotic cells. Apigenin reduced the mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance­associated proteins (MRPs) in MCF­7/ADR cells. Apigenin also downregulated the expression of P­gp. Apigenin reversed drug efflux from MCF­7/ADR cells, resulting in rhodamine 123 (Rho123) accumulation. Inhibition of drug resistance by apigenin is related to the suppression of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Apigenin decreased STAT3 activation (p­STAT3) and its nuclear translocation and inhibited the secretion of VEGF and MMP­9, which are STAT3 target genes. A STAT3 inhibitor, JAK inhibitor I and an HIF­1α inhibitor decreased cell growth in MCF­7 and MCF­7/ADR cells. Taken together, these results demonstrate that apigenin can overcome drug resistance.

Rubus coreanus Miquel extract causes apoptosis of doxorubicin-resistant NCI/ADR-RES ovarian cancer cells via JNK phosphorylation.

Cancer cells can acquire an anticancer, drug-resistant phenotype following chemotherapy, which is tightly linked to cancer malignancy and patient survival rates. Therefore, the identification of options to treat chemotherapy­resistant cancer cells is an urgent requirement. Rubus coreanus Miquel (RCM) has long been used as a source of food. In addition, it has been reported that RCM has effective functions against particular diseases, including cancer and inflammation. In the present study, it was demonstrated that RCM extract caused the apoptotic cell death of doxorubicin­resistant NCI/ADR­RES ovarian cancer cells by phosphorylating c­Jun N­terminal kinase (JNK). The RCM­mediated reduction of cell viability showed no synergism with doxorubicin. In addition, ellagic acid and quercetin, which are phytochemicals found in RCM, also caused apoptosis of the NCI/ADR­RES cells. In subsequent investigations of the RCM­altered signaling pathway, RCM extract, ellagic acid and quercetin were found to commonly induce the phosphorylation of JNK and AKT. Additionally, the inhibition of JNK with SP600125 repressed the apoptotic cell death induced by RCM extract, ellagic acid and quercetin, and the inhibition of JNK appeared to switch apoptosis to necrosis. JNK inhibition also reduced the phosphorylation of AKT, which was induced by RCM extract, ellagic acid and quercetin, suggesting that the phosphorylation of JNK is required for AKT phosphorylation in RCM­, ellagic acid­ or quercetin­induced apoptotic cell death. Therefore, the data obtained in the present study led to the conclusion that RCM caused apoptosis of doxorubicin­resistant NCI/ADR-RES ovarian cancer cells via JNK phosphorylation, and suggested that RCM may be effective in the treatment of chemotherapy­resistant cancer cells.

DSGOST inhibits tumor growth by blocking VEGF/VEGFR2-activated angiogenesis.

Tumor growth requires a process called angiogenesis, a new blood vessel formation from pre-existing vessels, as newly formed vessels provide tumor cells with oxygen and nutrition. Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang (DSGOST), one of traditional Chinese medicines, has been widely used in treatment of vessel diseases including Raynaud's syndrome in Northeast Asian countries including China, Japan and Korea. Therefore, we hypothesized that DSGOST might inhibit tumor growth by targeting newly formed vessels on the basis of its historical prescription. Here, we demonstrate that DSGOST inhibits tumor growth by inhibiting VEGF-induced angiogenesis. DSGOST inhibited VEGF-induced angiogenic abilities of endothelial cells in vitro and in vivo, which resulted from its inhibition of VEGF/VEGFR2 interaction. Furthermore, DSGOST attenuated pancreatic tumor growth in vivo by reducing angiogenic vessel numbers, while not affecting pancreatic tumor cell viability. Thus, our data conclude that DSGOST inhibits VEGF-induced tumor angiogenesis, suggesting a new indication for DSGOST in treatment of cancer.

SH003 represses tumor angiogenesis by blocking VEGF binding to VEGFR2.

Tumor angiogenesis is a key feature of cancer progression, because a tumor requires abundant oxygen and nutrition to grow. Here, we demonstrate that SH003, a mixed herbal extract containing Astragalus membranaceus (Am), Angelica gigas (Ag) and Trichosanthes Kirilowii Maximowicz (Tk), represses VEGF-induced tumor angiogenesis both in vitro and in vivo. SH003 inhibited VEGF-induced migration, invasion and tube formation in human umbilical vein endothelial cells (HUVEC) with no effect on the proliferation. SH003 reduced CD31-positive vessel numbers in tumor tissues and retarded tumor growth in our xenograft mouse tumor model, while SH003 did not affect pancreatic tumor cell viability. Consistently, SH003 inhibited VEGF-stimulated vascular permeability in ears and back skins. Moreover, SH003 inhibited VEGF-induced VEGFR2-dependent signaling by blocking VEGF binding to VEGFR2. Therefore, our data conclude that SH003 represses tumor angiogenesis by inhibiting VEGF-induced VEGFR2 activation, and suggest that SH003 may be useful for treating cancer.

SH003 selectively induces p73­dependent apoptosis in triple­negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a breast cancer subtype that has an aggressive phenotype, is highly metastatic, has limited treatment options and is associated with a poor prognosis. In addition, metastatic TNBC has no preferred standard chemotherapy due to resistance to anthracyclines and taxanes. The present study demonstrated that a herbal extract, SH003, reduced cell viability and induced apoptosis in TNBC without cell cytotoxicity. Cell viability was examined using trypan blue exclusion and colony formation assays, which revealed a decrease in the cell viability. Additionally, apoptosis was determined using flow cytometry and a sub­G1 assay, which revealed an increase in the proportion of cells in the sub­G1 phase. The present study investigated the anticancer effect of SH003 in the Hs578T, MDA­MB­231 and ZR­751 TNBC cell lines, and in the MCF7 and T47D non­TNBC cell lines. Western blot analysis revealed that the expression levels of poly­ADP­ribose polymerase (PARP) cleavage protein in cells treated with SH003 were increased dose­dependent manner, indicating that SH003 induced apoptosis via a caspase­dependent pathway. Pre­treatment with the caspase inhibitor Z­VAD reduced SH003­induced apoptosis was examined using trypan blue exclusion. Moreover, SH003 treatment enhanced the p73 levels in MDA­MB­231 cells but not in MCF7 cells. Transfection of p73 small interfering RNA (siRNA) in MDA­MB0231 cells revealed that the apoptotic cell death induced by SH003 was significantly impaired in comparison with scramble siRNA transfected MDA­MB­231 cells. This was examined using trypan blue exclusion and flow cytometry analysis (sub­G1). In addition, SH003 and paclitaxel exhibited synergistic anticancer effects on TNBC cells. The results indicate that SH003 exerts its anticancer effect via p73 protein induction and exhibits synergistic anticancer effects when combined with paclitaxel.

JNK1/2 Activation by an Extract from the Roots of Morus alba L. Reduces the Viability of Multidrug-Resistant MCF-7/Dox Cells by Inhibiting YB-1-Dependent MDR1 Expression.

Cancer cells acquire anticancer drug resistance during chemotherapy, which aggravates cancer disease. MDR1 encoded from multidrug resistance gene 1 mainly causes multidrug resistance phenotypes of different cancer cells. In this study, we demonstrate that JNK1/2 activation by an extract from the root of Morus alba L. (White mulberry) reduces doxorubicin-resistant MCF-7/Dox cell viability by inhibiting YB-1 regulation of MDR1 gene expression. When MCF-7 or MCF-7/Dox cells, where MDR1 is highly expressed were treated with an extract from roots or leaves of Morus alba L., respectively, the root extract from the mulberry (REM) but not the leaf extract (LEM) reduced cell viabilities of both MCF-7 and MCF-7/Dox cells, which was enhanced by cotreatment with doxorubicin. REM but not LEM further inhibited YB-1 nuclear translocation and its regulation of MDR1 gene expression. Moreover, REM promoted phosphorylation of c-Jun NH2-terminal kinase 1/2 (JNK1/2) and JNK1/2 inhibitor, SP600125 and rescued REM inhibition of both MDR1 expression and viabilities in MCF-7/Dox cells. Consistently, overexpression of JNK1, c-Jun, or c-Fos inhibited YB-1-dependent MDR1 expression and reduced viabilities in MCF-7/Dox cells. In conclusion, our data indicate that REM-activated JNK-cJun/c-Fos pathway decreases the viability of MCF-7/Dox cells by inhibiting YB-1-dependent MDR1 gene expression. Thus, we suggest that REM may be useful for treating multidrug-resistant cancer cells.

Autophagy Inhibition with Monensin Enhances Cell Cycle Arrest and Apoptosis Induced by mTOR or Epidermal Growth Factor Receptor Inhibitors in Lung Cancer Cells.

BACKGROUND: In cancer cells, autophagy is generally induced as a pro-survival mechanism in response to treatment-associated genotoxic and metabolic stress. Thus, concurrent autophagy inhibition can be expected to have a synergistic effect with chemotherapy on cancer cell death. Monensin, a polyether antibiotic, is known as an autophagy inhibitor, which interferes with the fusion of autophagosome and lysosome. There have been a few reports of its effect in combination with anticancer drugs. We performed this study to investigate whether erlotinib, an epidermal growth factor receptor inhibitor, or rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, is effective in combination therapy with monensin in non-small cell lung cancer cells. METHODS: NCI-H1299 cells were treated with rapamycin or erlotinib, with or without monensin pretreatment, and then subjected to growth inhibition assay, apoptosis analysis by flow cytometry, and cell cycle analysis on the basis of the DNA contents histogram. Finally, a Western blot analysis was done to examine the changes of proteins related to apoptosis and cell cycle control. RESULTS: Monensin synergistically increases growth inhibition and apoptosis induced by rapamycin or erlotinib. The number of cells in the sub-G1 phase increases noticeably after the combination treatment. Increase of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase, and decrease of anti-apoptotic proteins, bcl-2 and bcl-xL, are augmented by the combination treatment with monensin. The promoters of cell cycle progression, notch3 and skp2, decrease and p21, a cyclin-dependent kinase inhibitor, accumulates within the cell during this process. CONCLUSION: Our findings suggest that concurrent autophagy inhibition could have a role in lung cancer treatment.

Dual Inhibition of PI3K/Akt/mTOR Pathway and Role of Autophagy in Non-Small Cell Lung Cancer Cells.

BACKGROUND: The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit this pathway are currently under development for lung cancer treatment. In the present study, we have tested whether dual inhibition of PI3K/Akt/mTOR signaling can lead to enahnced antitumor effects. We have also examined the role of autophagy during this process. METHODS: We analyzed the combination effect of the mTOR inhibitor, temsirolimus, and the Akt inhibitor, GSK690693, on the survival of NCI-H460 and A549 non-small cell lung cancer cells. Cell proliferation was determined by MTT assay and apoptosis induction was evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Autophagy induction was also evaluated by acridine orange staining. Changes of apoptosis or autophagy-related proteins were evaluated by western blot analysis. RESULTS: Combination treatment with temsirolimus and GSK690693 caused synergistically increased cell death in NCI-H460 and A549 cells. This was attributable to increased induction of apoptosis. Caspase 3 activation and poly(ADP-ribose) polymerase cleavage accompanied these findings. Autophagy also increased and inhibition of autophagy resulted in increased cell death, suggesting its cytoprotective role during this process. CONCLUSION: Taken together, our results suggest that the combination of temsirolimus and GSK690693 could be a novel strategy for lung cancer therapy. Inhibition of autophagy could also be a promising method of enhancing the combination effect of these drugs.

Phytoestrogens induce apoptosis via extrinsic pathway, inhibiting nuclear factor-kappaB signaling in HER2-overexpressing breast cancer cells.

BACKGROUND: Phytoestrogens are known to prevent tumor induction. But their molecular mechanisms of action are largely unknown. This study aimed to examine the effect of genistein and quercetin on proliferation and apoptosis in HER2-expressing breast cancer cells. MATERIALS AND METHODS: The antiproliferative effects of phytoestrogens were tested by proliferation assays. Flow cytometry was performed to analyze the cell cycle. The effect of phytoestrogens on cell-signaling molecules was determined by Western blotting. RESULTS: Genistein and quercetin inhibited the proliferation of MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with an increase of subG(0)/G(1) apoptotic fractions. Genistein and quercetin induced extrinsic apoptosis pathway, up-regulating p53. Genistein and quercetin reduced the phosphorylation level of IκBα, and abrogated the nuclear translocation of p65 and its phosphorylation within the nucleus. CONCLUSION: Genistein and quercetin exert their antiproliferative activity by inhibiting NFκB signaling. Phytoestrogens could be potential useful compounds to prevent or treat HER2-overexpressing breast cancer.