Autophagy induction enhances tetrandrine-induced apoptosis via the AMPK/mTOR pathway in human bladder cancer cells

Abstract

Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra is a traditional Chinese medicine and exerts anticancer capacity in various types of cancers. Previous studies have shown that tetrandrine induces apoptosis in bladder cancer cells via activation of the caspase cascade. However, the underlying mechanism has not yet been reported. Autophagy is a cellular process involved in the degradation of broken proteins and aging organelles to maintain homeostasis. Recent studies indicate that autophagy is implicated in cancer therapy. Thus, we focused on the correlation between autophagy and apoptosis upon tetrandrine treatment in human bladder cancer cells. Firstly, our results observed a marked increase in autophagic double-membrane vacuoles and fluorescent puncta of red fluorescence protein-green fluorescence protein-LC3 (GRP-RFP-LC3) upon tetrandrine treatment, as evidenced by transmission electron microscopy and confocal fluorescence microscopy. Secondly, the expression of LC3-II was increased in tetrandrine-treated T24 and 5637 cells in a time- and concentration-dependent manner. Subsequently, downregulation of p62 and LC3 turnover assay further confirmed that tetrandrine induced autophagic flux in bladder cancer T24 and 5637 cells. Thirdly, the protein levels of phosphorylated-AMP-activated protein kinase (AMPK) and phosphorylated-acetyl-coenzyme A carboxylase (ACC) were upregulated in the tetrandrine-treated cells, while the mammalian target of rapamycin (mTOR)-related proteins were downregulated. Moreover, AICAR, a common AMPK activator, further increased the expression the LC3-II, while AMPK inhibitor compound C partially reversed the LC3-II protein levels in bladder cancer T24 cells. Finally, AICAR significantly reinforced the growth inhibition and apoptosis induction of tetrandrine in T24 and 5637 cells, while compound C had an opposite effect, suggesting that AMPK-mediated autophagy enhanced the cytotoxic and pro-apoptosis effect of tetrandrine in human bladder cancer cells. Taken together, the present study showed that tetrandrine induced autophagy in human bladder cancer cells by regulating the AMPK/mTOR signaling pathway, which contributed to the apoptosis induction by tetrandrine, indicating that tetrandrine may be a potential anticancer candidate for the treatment of bladder cancer, and autophagy may be a possible mechanism for cancer therapy.

Tetrandrine: a review of its anticancer potentials, clinical settings, pharmacokinetics and drug delivery systems

“…The induction of apoptosis by tetrandrine has been performed in many types of cancer cells, including pancreatic cancer cells, [ 95 ] leukaemia cells [ 101 ] and non‐small‐cell lung cancer (NSCLC) cells. [ 102 ] An apoptosis mediated by tetrandrine occurs mainly through the regulation of the following signalling pathways: (1) the AMPK/mTOR [ 60 ] ; (2) the PI3K/AKT [ 81 ] ; (3) the YAP [ 82 ] ; (4) the PI3K/AKT/mTOR [ 69 ] ; (5) the MAPKs [ 55 ] ; (6) the Wnt/β‐catenin [ 83 ] ; (7) the Wnt/cadherin [ 65 ] ; (8) the ERK/MAP kinase and ERK [ 84 ] ; (9) the PARP [ 41 ] ; (10) the caspases/beclin I/LC3‐I/II [ 61 ] ; (11) the caspase cascade and mitochondrial [ 59 ] ; (12) the death receptor pathway [ 64 ] , and any more.…”

“…Autophagy, a ‘self‐eating’ phenomenon, is a cellular process involved in the degradation of damaged proteins and aging organelles to maintain homeostasis [ 60 ] and is associated with cell death. [ 129 ] Research shows that tetrandrine induces autophagy in human bladder cancer T24 and 5637 cell lines by increasing the expression of LC3‐II, up‐regulating the protein levels of AMPK and ACC, and down‐regulating mTOR‐related proteins in a time‐ and concentration‐dependent manner via the regulation of the AMPK/mTOR signalling pathways.…”

“…These pharmacological studies indicated that tetrandrine has value in the prevention and treatment of multiple cancers, including colon cancer, [ 55–58 ] bladder cancer, [ 59,60 ] oral cancer, [ 61,62 ] prostate cancer, [ 63,64 ] ovarian cancer, [ 65 ] gastric cancer, [ 66,67 ] breast cancer, [ 68–70 ] pancreatic cancer, [ 70,71 ] osteosarcoma, [ 72 ] cervical cancer, [ 73 ] lung cancer, [ 74–76 ] liver cancer [ 77–79 ] and colorectal cancer, [ 80 ] both in vitro and in vivo . In addition, it also has revealed that tetrandrine displays significant anticancer activities mainly through the regulation of such signalling pathways as the AMP‐activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR), [ 60 ] the phosphoinositide‐3‐kinase (PI3K)/protein kinase B (AKT), [ 81 ] the Hippo/Yes‐ associated protein (YAP), [ 82 ] the PI3K/AKT/mTOR, [ 69 ] the p38 mitogen‐activated protein kinases (p38‐MAPKs), [ 55 ] the wingless (Wnt)/β‐catenin, [ 83 ] the Wnt/cadherin, [ 65 ] the extracellular signal‐regulated kinase (ERK) MAP kinase and ERK, [ 84 ] the poly (ADP ribose) polymerase (PARP), [ 41 ] the caspases/Becline I/LC3‐I/II [ 61 ] and the caspase cascade and mitochondrial pathways. [ 59 ] Additionally, several studies have reported that nano‐drug delivery systems could effectively encapsulate tetrandrine to improve therapeutic efficiency and reduce or restrain side effects and toxicity in cancer therapy.…”

Anticancer Effects of Green Tea and the Underlying Molecular Mechanisms in Bladder Cancer

“…This process is stimulated by various stimuli, including nutrients [ 71 , 72 ]. In fact, in bladder cancer cells, autophagy plays an important role in pathological processes and signaling pathways via external stimuli, including natural products [ 73 , 74 ]. With regard to the relationship between autophagy and GTPs, an in vitro study showed that the latter inhibited epirubicin-induced autophagy [ 75 ].…”

Combination therapy with protein kinase inhibitor H89 and Tetrandrine elicits enhanced synergistic antitumor efficacy

“…Surprisingly, H89/tetrandrine-induced cell death could not be completely reversed by the apoptosis inhibitor z-VAD-fmk, which implies that apoptosis was not the only contributor. Autophagy is involved in type-II programmed cell death, particularly in apoptosis-deficient cells, and may be exploited to suppress tumor growth [ 47 , 48 ]. Our results showed that H89/tetrandrine-induced cell death was moderately diminished by the autophagy inhibitor, which demonstrates the contribution of autophagy to cell death in response to treatment.…”