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Pachymic acid induces apoptosis via activating ROS-dependent JNK and ER stress pathways in lung cancer cells.

Ma J, Liu J, Lu C, Cai D - Cancer Cell Int. (2015)

Bottom Line: The in vivo efficacy of PA was measured using a NCI-H23 xenograft model in nude mice.Moreover, blockage of ROS production reversed PA-induced JNK and ER stress activation.Finally, PA inhibited the growth of NCI-H23 xenograft tumors without causing any host toxicity, and inhibited cell proliferation and induction of apoptosis of tumor cells in tumor xenograft tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Medicine, Zhongshan Hospital, Fudan University, Shanghai, 200032 China.

ABSTRACT

Background: Pachymic acid (PA), a lanostane-type triterpenoid from Poria cocos, has been reported to possess anti-emetic, anti-inflammatory, and anti-cancer properties. Nonetheless, the anti-tumor effect of PA in lung cancer cells remains unclear. Herein, we report the chemotherapeutic effects and underlying mechanisms of PA against human lung cancer.

Methods: The anti-proliferative ability of PA on lung cancer cells was assessed by MTT, colony formation and EdU proliferation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by annexin V/PI double-staining and the DNA ladder formation assays. The expressions of the apoptosis-related proteins were analysed by western blot. The in vivo efficacy of PA was measured using a NCI-H23 xenograft model in nude mice.

Results: PA exhibited anti-tumor effects in vitro accompanied by induction of G2/M phase arrest and apoptosis in NCI-H23 and NCI-H460 lung cancer cells. Mechanistically, our data showed that PA induced reactive oxygen species (ROS) production, resulting in the activation of both c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress apoptotic pathways in lung cancer cells. Moreover, blockage of ROS production reversed PA-induced JNK and ER stress activation. Finally, PA inhibited the growth of NCI-H23 xenograft tumors without causing any host toxicity, and inhibited cell proliferation and induction of apoptosis of tumor cells in tumor xenograft tissues.

Conclusions: In summary, our study demonstrates that PA induces apoptosis through activation of the JNK and ER stress pathways in human lung cancer cells. Our findings provide a rationale for the potential application of PA in lung cancer therapy.

No MeSH data available.


Related in: MedlinePlus

ROS generation mediates PA-activated apoptotic pathways. a NCI-H23 and NCI-H460 cells were treated with PA (20, 40 and 80 µM) for 24 h. The protein levels of p-JNK, p-ERK, p-p38, Bcl-2 and Bax were determined by western blot. b NCI-H23 and NCI-H460 cells were pre-treated with 20 µM JNK inhibitor (SP600125), p38 inhibitor (SB203580), ERK inhibitor (PD98059) for 1 h, or pre-treated with ROS inhibitor NAC (5 mM) for 2 h, then exposed to PA (80 µM) for 24 h. Cell viability was determined by MTT assay. c NCI-H23 and NCI-H460 cells were treated with PA (20, 40 and 80 µM) for 24 h. The protein levels of ATF4, ATF6, XBP-1 and CHOP were determined by western blot. d NCI-H23 and NCI-H460 cells were pre-treated with or without 5 mM NAC for 2 h before exposure to PA (80 μM) for 24 h. The protein levels of p-JNK, Bcl-2, Bax, ATF4, ATF6, XBP-1 and CHOP were determined by western blot. Data are presented as mean ± SD from three independent experiments. **P < 0.01 compared with the control group.
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Fig3: ROS generation mediates PA-activated apoptotic pathways. a NCI-H23 and NCI-H460 cells were treated with PA (20, 40 and 80 µM) for 24 h. The protein levels of p-JNK, p-ERK, p-p38, Bcl-2 and Bax were determined by western blot. b NCI-H23 and NCI-H460 cells were pre-treated with 20 µM JNK inhibitor (SP600125), p38 inhibitor (SB203580), ERK inhibitor (PD98059) for 1 h, or pre-treated with ROS inhibitor NAC (5 mM) for 2 h, then exposed to PA (80 µM) for 24 h. Cell viability was determined by MTT assay. c NCI-H23 and NCI-H460 cells were treated with PA (20, 40 and 80 µM) for 24 h. The protein levels of ATF4, ATF6, XBP-1 and CHOP were determined by western blot. d NCI-H23 and NCI-H460 cells were pre-treated with or without 5 mM NAC for 2 h before exposure to PA (80 μM) for 24 h. The protein levels of p-JNK, Bcl-2, Bax, ATF4, ATF6, XBP-1 and CHOP were determined by western blot. Data are presented as mean ± SD from three independent experiments. **P < 0.01 compared with the control group.

Mentions: To further investigate the underlying mechanisms involved in PA-induced effects on apoptosis in lung cancer cells. NCI-H23 and NCI-H460 cells were used for the subsequent studies. The mitogen-activated protein kinase (MAPK) pathway has been extensively evaluated in lung cancer, from the perspective of biomarkers of outcome and targets of therapy [14]. Therefore, we first focused on MAPK pathway. Surprisingly, we found that PA treatment significantly activated all of three pathways of MAPKs, including JNK, ERK, and p38 (Fig. 3a). We then determined the roles of JNK, ERK, and p38 in PA-induced cytotoxicity using specific small-molecule inhibitors. NCI-H23 and NCI-H460 cells were pre-treated with JNK inhibitor SP600125, ERK inhibitor PD98059, or p38 inhibitor SB203580, respectively for 1 h, then exposed to PA for 24 h. Interestingly, PD98059 or SB203580 did not alter the cell viability, but JNK inhibitor SP600125 partially attenuated PA-induced cytotoxicity, indicating that JNK activation was associated with PA-induced cell death (Fig. 3b). JNK is a well-known regulator in mitochondrial apoptotic pathway [15]. We therefore examined the effects of PA on Bcl-2 family proteins by western blot analysis, the results showed that Bcl-2 level was decreased, whereas Bax level was increased in a concentration-dependent manner (Fig. 3a). These results suggested that PA-induced NCI-H23 and NCI-H460 cells apoptosis is at least partly mediated by JNK-mitochondrial pathway.Fig. 3


Pachymic acid induces apoptosis via activating ROS-dependent JNK and ER stress pathways in lung cancer cells.

Ma J, Liu J, Lu C, Cai D - Cancer Cell Int. (2015)

ROS generation mediates PA-activated apoptotic pathways. a NCI-H23 and NCI-H460 cells were treated with PA (20, 40 and 80 µM) for 24 h. The protein levels of p-JNK, p-ERK, p-p38, Bcl-2 and Bax were determined by western blot. b NCI-H23 and NCI-H460 cells were pre-treated with 20 µM JNK inhibitor (SP600125), p38 inhibitor (SB203580), ERK inhibitor (PD98059) for 1 h, or pre-treated with ROS inhibitor NAC (5 mM) for 2 h, then exposed to PA (80 µM) for 24 h. Cell viability was determined by MTT assay. c NCI-H23 and NCI-H460 cells were treated with PA (20, 40 and 80 µM) for 24 h. The protein levels of ATF4, ATF6, XBP-1 and CHOP were determined by western blot. d NCI-H23 and NCI-H460 cells were pre-treated with or without 5 mM NAC for 2 h before exposure to PA (80 μM) for 24 h. The protein levels of p-JNK, Bcl-2, Bax, ATF4, ATF6, XBP-1 and CHOP were determined by western blot. Data are presented as mean ± SD from three independent experiments. **P < 0.01 compared with the control group.
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Fig3: ROS generation mediates PA-activated apoptotic pathways. a NCI-H23 and NCI-H460 cells were treated with PA (20, 40 and 80 µM) for 24 h. The protein levels of p-JNK, p-ERK, p-p38, Bcl-2 and Bax were determined by western blot. b NCI-H23 and NCI-H460 cells were pre-treated with 20 µM JNK inhibitor (SP600125), p38 inhibitor (SB203580), ERK inhibitor (PD98059) for 1 h, or pre-treated with ROS inhibitor NAC (5 mM) for 2 h, then exposed to PA (80 µM) for 24 h. Cell viability was determined by MTT assay. c NCI-H23 and NCI-H460 cells were treated with PA (20, 40 and 80 µM) for 24 h. The protein levels of ATF4, ATF6, XBP-1 and CHOP were determined by western blot. d NCI-H23 and NCI-H460 cells were pre-treated with or without 5 mM NAC for 2 h before exposure to PA (80 μM) for 24 h. The protein levels of p-JNK, Bcl-2, Bax, ATF4, ATF6, XBP-1 and CHOP were determined by western blot. Data are presented as mean ± SD from three independent experiments. **P < 0.01 compared with the control group.
Mentions: To further investigate the underlying mechanisms involved in PA-induced effects on apoptosis in lung cancer cells. NCI-H23 and NCI-H460 cells were used for the subsequent studies. The mitogen-activated protein kinase (MAPK) pathway has been extensively evaluated in lung cancer, from the perspective of biomarkers of outcome and targets of therapy [14]. Therefore, we first focused on MAPK pathway. Surprisingly, we found that PA treatment significantly activated all of three pathways of MAPKs, including JNK, ERK, and p38 (Fig. 3a). We then determined the roles of JNK, ERK, and p38 in PA-induced cytotoxicity using specific small-molecule inhibitors. NCI-H23 and NCI-H460 cells were pre-treated with JNK inhibitor SP600125, ERK inhibitor PD98059, or p38 inhibitor SB203580, respectively for 1 h, then exposed to PA for 24 h. Interestingly, PD98059 or SB203580 did not alter the cell viability, but JNK inhibitor SP600125 partially attenuated PA-induced cytotoxicity, indicating that JNK activation was associated with PA-induced cell death (Fig. 3b). JNK is a well-known regulator in mitochondrial apoptotic pathway [15]. We therefore examined the effects of PA on Bcl-2 family proteins by western blot analysis, the results showed that Bcl-2 level was decreased, whereas Bax level was increased in a concentration-dependent manner (Fig. 3a). These results suggested that PA-induced NCI-H23 and NCI-H460 cells apoptosis is at least partly mediated by JNK-mitochondrial pathway.Fig. 3

Bottom Line: The in vivo efficacy of PA was measured using a NCI-H23 xenograft model in nude mice.Moreover, blockage of ROS production reversed PA-induced JNK and ER stress activation.Finally, PA inhibited the growth of NCI-H23 xenograft tumors without causing any host toxicity, and inhibited cell proliferation and induction of apoptosis of tumor cells in tumor xenograft tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Medicine, Zhongshan Hospital, Fudan University, Shanghai, 200032 China.

ABSTRACT

Background: Pachymic acid (PA), a lanostane-type triterpenoid from Poria cocos, has been reported to possess anti-emetic, anti-inflammatory, and anti-cancer properties. Nonetheless, the anti-tumor effect of PA in lung cancer cells remains unclear. Herein, we report the chemotherapeutic effects and underlying mechanisms of PA against human lung cancer.

Methods: The anti-proliferative ability of PA on lung cancer cells was assessed by MTT, colony formation and EdU proliferation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by annexin V/PI double-staining and the DNA ladder formation assays. The expressions of the apoptosis-related proteins were analysed by western blot. The in vivo efficacy of PA was measured using a NCI-H23 xenograft model in nude mice.

Results: PA exhibited anti-tumor effects in vitro accompanied by induction of G2/M phase arrest and apoptosis in NCI-H23 and NCI-H460 lung cancer cells. Mechanistically, our data showed that PA induced reactive oxygen species (ROS) production, resulting in the activation of both c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress apoptotic pathways in lung cancer cells. Moreover, blockage of ROS production reversed PA-induced JNK and ER stress activation. Finally, PA inhibited the growth of NCI-H23 xenograft tumors without causing any host toxicity, and inhibited cell proliferation and induction of apoptosis of tumor cells in tumor xenograft tissues.

Conclusions: In summary, our study demonstrates that PA induces apoptosis through activation of the JNK and ER stress pathways in human lung cancer cells. Our findings provide a rationale for the potential application of PA in lung cancer therapy.

No MeSH data available.


Related in: MedlinePlus