Involvement of Autophagy in Differential Cytotoxicity of Pterostilbene on Human Bladder Cancer Cells and Immortalized Human Uroepithelial Cells to FDA‐Approved Anticancer Drugs

Abstract

Pterostilbene, a dimethylether analog of resveratrol, can be found in grape vines and blueberries. Both of which are polyphenol phytoalexins and have many physiological and pharmacological similarities, such as antineoplastic effects. Bladder cancer is the ninth most common cancer worldwide. Combination of cisplatin and gemcitabine is the standard first‐line treatment for patients with locally advanced and metastatic bladder cancer. Carboplatin plus gemcitabine has been used for patients considered unfit to receive cisplatin due to its major side effect of nephrotoxicity. Autophagy is involved in the degradation and recycling of proteins and intracellular components in response to starvation and stress. Four faces of autophagy have been characterized, including cytoprotective (resistance to therapy), cytotoxic (promoting cell death), cytostatic (associated with senescence), and nonprotective (no influence in sensitivity to therapy). Here we reveal that pterostilbene exhibited better cytotoxicity and induced stronger autophagy than resveratrol on grade III human bladder cancer T24 cells. Coadministration of pterostilbene elevated cytotoxicity of T24 cells and immortalized human uroepithelial E7 cells to gemcitabine plus cisplatin/carboplatin, producing a synergy effect on T24 cells and caused an antagonism effect on E7 cells. Flow cytometric analysis on T24 cells revealed that addition of pterostilbene neither elevated apoptosis nor induced cell cycle arrest, but greatly increased the cells with acidic vesicular organelles, representing the formation of autolysosomes at the later stage of autophagic flux. Expression of cleaved caspase 3 (at the later stage of apoptosis) and LC3‐II (at the early stage of autophagic process) using Western blot confirmed that addition of pterostilbene promoted autophagy but not apoptosis. No induction of autophagy was observed in E7 cells. Pterostilbene‐induced autophagy on T24 cells was paralleled with inhibition of class I PI3K/mTOR/p70S6K and activation of MEK/ERK and class III PI3K pathways, but was not concurrent with increase of Atg12‐Atg5. Furthermore, pterostilbene elevated the induction of cell senescence in gemcitabine plus cisplatin/carboplatin‐treated T24 cells determined using senescence‐associated β‐galactosidase activity. No cell senescence was observed in E7 cells. Pterostilbene‐induced cell senescence on T24 cells was paralleled with increased pan‐Ras and decreased phospho‐pRB expression. Administration of autophagy inhibitor to class III PI3K (3‐methyladenine) or MEK (U0126) suppressed pterostilbene‐induced autophagy and reversed pterostilbene‐enhanced cytotoxicity, but did not affect pterostilbene‐elevated cell senescence or apoptosis on T24 cells. Our data demonstrate that pterostilbene‐produced synergistic biosensitivity of T24 cells to gemcitabine plus cisplatin/carboplatin proceeded via induction of cytotoxic form of autophagic flux, suggesting a combination of pterostilbene with the FDA‐approved anticancer drugs for patients with bladder cancer.Support or Funding InformationSupported in part by the National Science Council, Taiwan, No. NSC 101‐2313‐B‐003‐002‐MY3.