Kaempferol induces apoptosis through the MAPK pathway and regulates JNK-mediated autophagy in MC-3 cells.

This study sought to determine the anticancer effect of kaempferol, a glycone-type flavonoid glycoside with various pharmacological benefits, on human oral cancer MC-3 cells. In vitro studies comprised a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, annexin V and propidium iodide staining, western blotting analysis, and acridine orange staining, while the in vivo studies entailed a xenograft model, hematoxylin and eosin staining, and TdT-mediated dUTP-biotin nick end labelling. In vitro, kaempferol reduced the rate of survival of MC-3 cells, mediated intrinsic apoptosis, increased the number of acidic vesicular organelles, and altered the expression of autophagy-related proteins. Further, treatment with the autophagy inhibitors revealed that the induced autophagy had a cytoprotective effect on apoptosis in kaempferol-treated MC-3 cells. Kaempferol also decreased the expression of phosphorylated extracellular signal-regulated kinase and increased that of phosphorylated c-Jun N-terminal kinase (p-JNK) and phosphorylated p38 kinase in MC-3 cells, suggesting the occurrence of mitogen-activated protein kinase-mediated apoptosis and JNK-mediated autophagy. In vivo, kaempferol reduced tumor growth inducing apoptosis and autophagy. These results showed that kaempferol has the potential use as an adjunctive agent in treating oral cancer.

Platycodin D induces apoptosis via regulating MAPK pathway and promotes autophagy in colon cancer cell.

Platycodin D (PD) is the main component of triterpene saponins found in Platycodi radix. In this study, we observed a decrease in cell viability, an increase in apoptotic bodies, and an increase in the rate of apoptosis. Also, we observed an increase in cleaved PARP and Bax, a decrease in Bcl-2, and p-ERK, and an increase in p-p38 and p-JNK. Furthermore, a change in cell viability and the expression of p-p38, Bax, and Bcl-2 using the p38 inhibitor revealed a decrease in p-p38 and Bax and an increase in Bcl-2 in the inhibitor treatment group. In addition, we observed an increase in vacuole formation through morphological changes and an increase in acidic vesicular organelles (AVOs). We also observed an increase in the expression of beclin 1, LC 3-I, and -II. There was no significant decrease in cell viability in the group treated with 3-MA, but a decrease in cell viability was noted in the group treated with HCQ. HCQ treatment resulted in an increase in Bax and a decrease in Bcl-2. These findings reveal that in HT-29 colon cancer cells, PD induces apoptosis through the MAPK pathway, thereby exerting anticancer effects. Moreover, autophagy caused by PD inhibits apoptosis by protecting the cells.

Dendropanax morbiferus H. Lév. Leaf Extract Inhibits the Proliferation of MDA-MB-231 Breast Cancer Cells and Induces Apoptosis via the MAPK Pathway In Vitro and In Vivo.

BACKGROUND/AIM: The toxic side effects of therapies against breast cancer can affect the quality of life of patients, necessitating the use of naturally-derived therapeutics. Here, we investigated the effects of Dendropanax morbiferus H. Lév. leaf (DPL) extract on breast cancer cells in vitro and in vivo to assess its anticancer potential. MATERIALS AND METHODS: MDA-MB-231 breast cancer cells were treated with DPL, and the in vitro effect of DPL on the cells was evaluated through an MTT assay, DAPI staining, annexin V/propidium iodide double staining, and western blotting. The in vivo effects of DPL were measured through the MDA-MB-231 tumor xenograft mouse model. A TUNEL assay and immunohistochemistry were used to determine the extent of apoptosis and p-p38 expression in tumor tissues, respectively. RESULTS: DPL treatment significantly suppressed cell viability in a concentration-dependent manner. Furthermore, DPL treatment resulted in increased apoptotic body formation, apoptosis rate, cleaved poly (ADP-ribose) polymerase and B-cell lymphoma 2 (Bcl-2)-associated X protein levels, phosphorylation of mitogen-activated protein kinase (MAPK) pathway proteins, and decreased Bcl-2 levels. In addition, the antitumor effect in vivo was confirmed through the xenograft model, where decreased tumor volume and weight following DPL administration were observed. Further, apoptosis and increased p-p38 levels in tumor tissues were observed, and no pathological abnormalities were found in the liver or kidney. CONCLUSION: DPL inhibits proliferation through MAPK-mediated apoptosis in breast cancer cells and tumors, suggesting the potential of DPL as a natural therapeutic agent for breast cancer.

Chrysin Induces Apoptosis via the MAPK Pathway and Regulates ERK/mTOR-Mediated Autophagy in MC-3 Cells.

Chrysin is a flavonoid found abundantly in substances, such as honey and phytochemicals, and is known to exhibit anticancer effects against various cancer cells. Nevertheless, the anticancer effect of chrysin against oral cancer has not yet been verified. Furthermore, the mechanism underlying autophagy is yet to be clearly elucidated. Thus, this study investigated chrysin-mediated apoptosis and autophagy in human mucoepidermoid carcinoma (MC-3) cells. The change in MC-3 cell viability was examined using a 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide cell viability assay, as well as 40,6-diamidino-2-phenylindole, annexin V, and propidium iodide staining. Western blotting was used to analyze the proteins related to apoptosis and the mitogen-activated protein kinase (MAPK) pathway. In addition, the presence or absence of autophagy and changes in the expression of related proteins were investigated using acridine orange staining and Western blot. The results suggested that chrysin induced apoptosis and autophagy in MC-3 oral cancer cells via the MAPK/extracellular signal-regulated kinase pathway. Moreover, the induced autophagy exerted a cytoprotective effect against apoptosis. Thus, the further reduced cell viability due to autophagy as well as apoptosis induction highlight therapeutic potential of chrysin for oral cancer.

Myricetin induces apoptosis and autophagy in human gastric cancer cells through inhibition of the PI3K/Akt/mTOR pathway.

Myricetin, a natural flavonoid present in berries, nuts, and green tea, is well-known for its anticancer properties. Even though several previous studies have reported the anticancer effects induced by myricetin, these effects have not yet been confirmed in the adenocarcinoma gastric cell line (AGS). Moreover, the exact mechanisms of myricetin-induced apoptosis and autophagy have not been clearly identified either. Therefore, in this study, we aimed to examine the role of myricetin in inducing apoptosis and autophagy in AGS gastric cancer cells. First, the survival rate of AGS gastric cancer cells was assessed using the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell viability assay. Thereafter, the rate of apoptosis was analyzed using4',6-diamidino-2-phenylindole (DAPI) staining as well as annexin V and propidium iodide (PI) staining, and the expression of the proteins associated with apoptosis, PI3K/Akt/mTOR pathway, and autophagy was examined by western blotting. We observed that myricetin reduced the survival rate of AGS gastric cancer cells by inhibiting the PI3K/Akt/mTOR pathway, thereby inducing apoptosis and autophagy. Similar results were also obtained in vivo, and tumor growth was inhibited. Therefore, in the AGS gastric cancer cells, myricetin seems to inhibit the PI3K/Akt/mTOR pathway, which in turn leads to apoptosis in vitroand in vivo, cell-protective autophagy, as well as inhibition of cancer cell proliferation. These results indicate the potential of myricetin as a natural anticancer agent.

Myricetin induces apoptosis through the MAPK pathway and regulates JNK­mediated autophagy in SK­BR­3 cells.

Myricetin, a flavonoid found in fruits and vegetables, is known to have antioxidant and anticancer effects. However, the anticancer effects of myricetin on SK­BR­3 human breast cancer cells have not been elucidated. In the present study, the anticancer effects of myricetin were confirmed in human breast cancer SK­BR­3 cells. As the concentration of myricetin increased, the cell viability decreased. DAPI (4',6­diamidino­2­phenylindole) and Annexin V/PI staining also revealed a significant increase in apoptotic bodies and apoptosis. Western blot analysis was performed to confirm the myricetin­induced expression of apoptosis­related proteins. The levels of cleaved PARP and Bax proteins were increased, and that of Bcl­2 was decreased. The levels of proteins in the mitogen­activated protein kinase (MAPK) pathway were examined to confirm the mechanism of myricetin­induced apoptosis, and it was found that the expression levels of phosphorylated c­Jun N­terminal kinase (p­JNK) and phosphorylated mitogen­activated protein kinases (p­p38) were increased, whereas that of phosphorylated extracellular­regulated kinase (p­ERK) was decreased. It was also demonstrated that myricetin induced autophagy by promoting autophagy­related proteins such as microtubule­associated protein 1A/1B­light chain 3 (LC 3) and beclin 1. In addition, 3­methyladenine (3­MA) was used to evaluate the association between cell viability and autophagy in cells treated with myricetin. The results showed that simultaneous treatment with 3­MA and myricetin promoted the apoptosis of breast cancer cells. Furthermore, treatment with a JNK inhibitor reduced cell viability, promoted Bax expression, and reduced the expression of p­JNK, Bcl­2, and LC 3­II/I. These results suggest that myricetin induces apoptosis via the MAPK pathway and regulates JNK­mediated autophagy in SK­BR­3 cells. In conclusion, myricetin shows potential as a natural anticancer agent in SK­BR­3 cells.

Anticancer effects of oleanolic acid on human melanoma cells.

Owing to the ineffectiveness of the currently used therapies against melanoma, there has been a shift in focus toward alternative therapies involving the use of natural compounds. This study assessed the anticancer effects of oleanolic acid (OA) and its ability to induce apoptosis in A375SM and A375P melanoma cells in vivo. Compared to the control group, viability of A375P and A375SM cells decreased following OA treatment. In OA-treated A375SM and A375P cells, 4',6-diamidino-2-phenylindole staining showed an increase in the apoptotic body, and flow cytometry revealed increased number of apoptotic cells compared to that in the control group. OA-treated A375SM cells exhibited an increased expression of the apoptotic proteins, cleaved poly (ADP-ribose) polymerase (PARP) and B-cell lymphoma (Bcl)-2-associated X protein (Bax) as well as decreased expression of the antiapoptotic protein Bcl-2 compared to that in the control group. In OA-treated A375P cells, expression patterns of cleaved PARP and Bcl-2 were similar to those in OA-treated A375SM cells; however, no difference was reported in the expression of Bax compared to that in the control group. Additionally, OA-treated melanoma cells showed decreased expression of phospho-nuclear factor-κB (p-NF-κB), phospho-inhibitor of nuclear factor-κBα (p-IκBα), and phospho-IκB kinase αß than that in the control group. Moreover, immunohistochemistry showed a comparatively decreased level of p-NF-κB in the OA-treated group than that in the control group. Xenograft analysis confirmed the in vivo anticancer effects of OA against A375SM cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay revealed an increased number of TUNEL-positive cells in the OA-treated group compared to that in the control group. In conclusion, the study results suggest that OA induces apoptosis of A375SM and A375P cells in vitro and apoptosis of A375SM cells in vivo. Furthermore, the in vitro and in vivo anticancer effects were mediated by the NF-κB pathway.

Silymarin inhibits proliferation of human breast cancer cells via regulation of the MAPK signaling pathway and induction of apoptosis.

Silymarin is a purified mixture of four isomeric flavonoids extracted from the seeds and fruit of the milk thistle plant, Silybum marianus (L.). Silymarin exhibits a wide variety of biological effects and is commonly used in traditional medicine. Therefore, the anticancer effects of silymarin on human breast cancer cells were investigated to determine its pharmacological mechanisms in vitro and in vivo. The viability and proliferation of MDA-MB- 231 and MCF-7 breast cancer cells were investigated using MTT and wound healing assays. Silymarin decreased the viability and proliferation of MDA-MB-231 and MCF-7 cells in a concentration-dependent manner. The number of apoptotic bodies, as shown by DAPI staining, was increased in a concentration-dependent manner, indicating that silymarin induces apoptosis. Additionally, changes in the expression levels of apoptosis-related proteins were demonstrated in human breast cancer cells using western blotting. Silymarin increased the levels of Bax, cleaved poly-ADP ribose polymerase, cleaved caspase-9 and phosphorylated (p-)JNK, and decreased the levels of Bcl-2, p-P38 and p-ERK1/2. Furthermore, the inhibitory effects of silymarin on MCF-7 tumor growth were investigated. In mice treated with silymarin for 3 weeks (25 and 50 mg/kg), MCF-7 tumor growth was inhibited without organ toxicity. In MCF-7 tumors, silymarin induced apoptosis and decreased p-ERK1/2 levels, as assessed using a TUNEL assay and immunohistochemistry. These results indicated that silymarin inhibited breast cancer cell proliferation both in vitro and in vivo by modulating the MAPK signaling pathway. Therefore, silymarin may potentially be used as a chemo-preventive or therapeutic agent.

Shikonin inhibits proliferation of melanoma cells by MAPK pathway-mediated induction of apoptosis.

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, exhibits pharmacological effects against inflammation, ulcers, infections, and tumors. In the present study, we aimed to investigate the antitumor effects of shikonin on the human melanoma cell line, A375SM, and in an in vivo mouse xenograft model. We examined the anticancer effects of shikonin by in vitro experiments (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 4',6-diamidino-2-phenylindole (DAPI) stain, annexin V/ propidium iodide (PI) stain, and protein analysis of apoptosis and mitogen-activated protein kinase (MAPK) pathways). Further, the anticancer effect in vivo was confirmed through a xenograft model. Our results showed that shikonin inhibited the proliferation of melanoma cells in a dose-dependent manner. In addition, shikonin significantly increased nucleus and chromatin condensation and early/late apoptosis. Shikonin also increased the pro-apoptotic proteins and decreased the anti-apoptotic proteins. Additionally, shikonin was overexpressed in MAPK pathways. Investigation of the effects of shikonin in a mouse xenograft model not only showed decreased A375SM tumor volume but also increased apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, pathologic changes were not observed in the liver and kidney of mice. Collectively, the study indicated that shikonin inhibited the proliferation of the human melanoma cells by inducing apoptosis, mediated by MAPK pathway and that it is a potential candidate for an anticancer drug against melanoma cancer.

Apigenin induces apoptosis by regulating Akt and MAPK pathways in human melanoma cell A375SM.

Apigenin, an aromatic compound, exhibits antioxidant, anti­inflammatory and anti­viral effects. The present study aimed to investigate the effects of apigenin on cell proliferation and apoptosis of human melanoma cells A375P and A375SM. Therefore, melanoma cells were treated with apigenin to determine its anti­proliferative and survival effects, using wound healing and MTT assays. The results revealed that melanoma cell viability was decreased in a dose­dependent manner. Furthermore, chromatin condensation, indicating apoptosis, was significantly increased in a dose­dependent manner, as demonstrated by DAPI staining. In addition, increased apoptosis rate following treatment with apigenin was confirmed by Annexin V­propidium iodide staining. The changes in the expression levels of apoptosis­related proteins in A375P and A375SM melanoma cells were subsequently detected using western blot analysis. The results demonstrated that the protein expression levels of Bcl­2 were decreased, whereas those of Bax, cleaved poly ADP­ribose polymerase, cleaved caspase­9 and p53 were upregulated in a dose­dependent manner in apigenin­treated cells compared with those noted in untreated cells. In addition, in apigenin­treated A375P cells, phosphorylated (p)­p38 was upregulated and p­extracellular signal­regulated kinase (ERK), p­c­Jun N­terminal kinase (JNK) and p­protein kinase B (Akt) were downregulated. However, in A375SM cells, apigenin treatment increased p­ERK and p­JNK and decreased p­p38 and p­Akt protein expression levels. Subsequently, the inhibitory effect of apigenin on tumor growth was investigated in vivo. Tumor volume was significantly reduced in the 25 and 50 mg/kg apigenin­treated groups compared with the control group. Additionally, a TUNEL assay was performed to detect apoptotic cells. Immunohistochemical staining also revealed elevated p­ERK expression in the apigenin­treated group compared with the control group. Overall, the findings of the present study indicated that apigenin attenuated the growth of A375SM melanoma cells by inducing apoptosis via regulating the Akt and mitogen­activated protein kinase signaling pathways.

Silymarin induces inhibition of growth and apoptosis through modulation of the MAPK signaling pathway in AGS human gastric cancer cells.

Apoptosis is regarded as a therapeutic target because it is typically disturbed in human cancer. Silymarin from milk thistle (Silybum marianum) has been reported to exhibit anticancer properties via regulation of apoptosis as well as anti­inflammatory, antioxidant and hepatoprotective effects. In the present study, the effects of silymarin on the inhibition of proliferation and apoptosis were examined in human gastric cancer cells. The viability of AGS human gastric cancer cells was assessed by MTT assay. The migration of AGS cells was investigated by wound healing assay. Silymarin was revealed to significantly decrease viability and migration of AGS cells in a concentration­dependent manner. In addition, the number of apoptotic bodies and the rate of apoptosis were increased in a dose­dependent manner as determined by DAPI staining and Annexin V/propidium iodide double staining. The changes in the expression of silymarin­induced apoptosis proteins were investigated in human gastric cancer cells by western blotting analysis. Silymarin increased the expression of Bax, phosphorylated (p)­JNK and p­p38, and cleaved poly­ADP ribose polymerase, and decreased the levels of Bcl­2 and p­ERK1/2 in a concentration­dependent manner. The in vivo tumor growth inhibitory effect of silymarin was investigated. Silymarin (100 mg/kg) significantly decreased the AGS tumor volume and increased apoptosis, as assessed by the TUNEL assay, confirming its tumor­inhibitory effect. Immunohistochemical staining revealed elevated expression of p­JNK and p­p38 as well as reduced expression of p­ERK1/2 associated with silymarin­treatment. Silymarin was revealed to reduce tumor growth through inhibition of p­ERK and activation of p­p38 and p­JNK in human gastric cancer cells. These results indicated that silymarin has potential for development as a cancer therapeutic due to its growth inhibitory effects and induction of apoptosis in human gastric cancer cells.

Antitumor and apoptotic effects of quercetin on human melanoma cells involving JNK/P38 MAPK signaling activation.

In this study, we investigated whether Quercetin has anti-cancer effects on A375SM and A375P human melanoma cells. Cell viability was assessed using an MTT assay. The proliferation of melanoma cells was measured by a wound-healing assay. Quercetin significantly decreased viability and proliferation of A375SM cells in a concentration-dependent manner. However, quercetin had no effect on A375P cells. DAPI staining showed increased chromatin condensation in a concentration-dependent manner, indicating apoptosis. Flow cytometric analysis indicated that quercetin suppressed the viability of A375SM cells by inducing apoptosis. Expression of quercetin-induced apoptosis proteins was investigated by Western blot analysis. Quercetin increased the expression of Bax, phospho-JNK, phospho-p38 and phospho-ERK1/2, cleaved poly-ADP ribose polymerase and decreased Bcl-2 in a concentration-dependent manner. We also investigated the in vivo tumor-growth inhibitory effect of quercetin. Quercetin (at 50 and 100 mg/kg) significantly decreased the A375SM tumor volume compared to the control group and increased apoptosis as assessed by the TUNEL assay. Immunohistochemistry staining revealed that the level of phosphor-JNK and phosphor-p38 increased in the quercetin-treated mice. These results indicate that quercetin inhibited the growth of A375SM melanoma cells through apoptosis and thus can be regarded as a new and effective chemo-preventive or therapeutic agent.

Antitumor and Apoptosis-inducing Effects of Piperine on Human Melanoma Cells.

BACKGROUND/AIM: Piperine is a major pungent alkaloid present in black pepper (Piper nigrum L). This study investigated the potential anticancer effects of piperine on human melanoma cells and explored the potential pharmacological mechanisms in vitro and in vivo. MATERIALS AND METHODS: Studies were performed using the MTT assay, 4',6-diamidino-2-phenylindole (DAPI) staining, western blotting, a xenograft model, the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and immunohistochemistry. RESULTS: Piperine inhibited the growth of melanoma cells. Several apoptotic events were observed following treatment, as revealed by DAPI staining. Piperine increased the expression of BCL2-associated X, apoptosis regulator (BAX), cleaved poly(ADP-ribose)polymerase, cleaved caspase-9, phospho-c-Jun N-terminal kinase and phospho-p38, and reduced that of B-cell lymphoma 2 (BCL2), X-chromosome-linked inhibitor of apoptosis, and phospho-extracellular signal-regulated protein kinase (ERK1/2) in a concentration-dependent manner. Treatment of mice for 4 weeks with piperine inhibited tumor growth without apparent toxicity. Piperine increased the expression of apoptotic cells and cleaved-caspase-3 protein and reduced the expression of phospho-ERK1/2 protein in melanoma tumors. CONCLUSION: Piperine suppressed the growth of human melanoma cells by the induction of apoptosis via the inhibition of tumor growth of human melanoma cells and tumor xenograft models.

Anti­inflammatory effects of Dendropanax morbifera in lipopolysaccharide­stimulated RAW264.7 macrophages and in an animal model of atopic dermatitis.

Dendropanax morbifera (D. morbifera), known as Dendro, means 'omnipotent drug' (Panax), and has been called the panacea tree. Various studies on D. morbifera are currently ongoing, aiming to determine its medicinal uses. The present study investigated the anti­inflammatory effects and underlying mechanism of a natural extract of D. morbifera leaves (DPL) in lipopolysaccharide (LPS)­stimulated RAW264.7 macrophages. In the present study, the following assays and models were used: MTT assay, nitric oxide (NO) assay, western blotting, ELISA and mouse models of atopic dermatitis. DPL extract markedly reduced the production of NO, inducible NO synthase and interleukin­6, as well as the nuclear translocation of nuclear factor­κB (NF­κB). Additionally, the LPS­induced activation of extracellular signal­regulated kinase 1/2 (ERK1/2), P38 and c­Jun N­terminal kinase (JNK) was suppressed by DPL extract. Taken together, these results indicate that NF­κB, ERK1/2, P38 and JNK may be potential molecular targets of DPL extract in the LPS­induced inflammatory response. Subsequently, the present study investigated the effects of DPL extract in a 2,4­dinitrochlorobenzene­induced atopic dermatitis mouse model. Ear thickness, serum immunoglobulin E levels and histological analysis revealed that the DPL extract was effective in attenuating the inflammatory response. These results indicate that DPL extract has anti­inflammatory potential and may be developed as a botanical drug to treat atopic dermatitis.