Cytisine induces endoplasmic reticulum stress caused by calcium overload in HepG2 cells. Yu Lei,Jiang Bo,Chen Zhifeng,Wang Xin,Shang Dongxue,Zhang Xiaopo,Sun Yongxue,Yang Jinghui,Ji Yubin Oncology reports Cytisine, a quinolizidine alkaloid, is one of the major bioactive components found in the small tree Sophora Alopecuraides L., and is a traditional Chinese medicine that is used for treating hepatitis and liver cancer. In the 1960s, quinolizidine alkaloids were reported to exhibit inhibitory effects on tumour cell proliferation in several types of cancer cells. However, few studies have investigated the effect of cytisine on liver cancer. Our team confirmed that cytisine induced apoptosis in HepG2 cells via a mitochondrial pathway. The primary aim of the present study was to evaluate the endoplasmic reticulum (ER) stress caused by calcium overload in cytisine‑induced apoptosis in HepG2 cells and the molecular mechanisms of this phenomenon. In addition, the present study was undertaken to evaluate the expression of α7‑nAChR when apoptosis was induced by cytisine in HepG2 cells. In the present study, transmission electron microscopy was used to observe the morphological appearance of HepG2 cells. The apoptosis of the cells with cytoplasmic vacuolization was significant under electron microscopy. Apoptotic bodies, the expansion of the ER, and swelling of mitochondria were observed in the HepG2 cells after cytisine treatment. Flow cytometric analysis demonstrated that the apoptosis rate of HepG2 cells was upregulated. In addition, the intracellular calcium concentration was detected by laser confocal fluorescence microscopy. The laser confocal fluorescence microscopy showed that the calcium concentration was increased in a dose‑dependent manner. The activity of caspase‑4 was evaluated by an enzyme‑linked analyser, and the expression levels of CHOP, JNK, p‑JNK and α7‑nAChR were assessed via western blot analysis. In the present study, we observed that cytisine induced ER stress‑inducing factors and CHOP and p‑JNK1/2 protein expression, and it increased the JNK protein expression in the HepG2 cells. Furthermore, α7‑nAChR protein expression was promoted in a dose‑dependent manner after cytisine treatment. These findings suggest that cytisine induced the ER stress‑mediated apoptotic pathway via activation of CHOP, JNK and caspase‑4 in HepG2 cells, and cytisine is a potential new target compound for nAchRs (nicotinic acetylcholine receptors) to treat liver cancer. 10.3892/or.2018.6200

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis. Zhang Jintao,Yi Man,Zha Longying,Chen Siqiang,Li Zhijia,Li Cheng,Gong Mingxing,Deng Hong,Chu Xinwei,Chen Jiehua,Zhang Zheqing,Mao Limei,Sun Suxia PloS one PURPOSE:Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells. METHODS:Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot. RESULTS:Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced. DISCUSSION:Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid. 10.1371/journal.pone.0147218

Cisplatin Induces Apoptosis Through the Endoplasmic Reticulum-mediated, Calpain 1 Pathway in Triple-negative Breast Cancer Cells. Al-Bahlani Shadia M,Al-Bulushi Khadija H,Al-Alawi Zaina M,Al-Abri Nadia Y,Al-Hadidi Zuweina R,Al-Rawahi Shaikha S Clinical breast cancer BACKGROUND:Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer (TNBC) is an aggressive type that can be treated using platinum-based chemotherapy such as cisplatin (cis-diamminedichloroplatinum II). Although the calpain protein is essential in many cellular processes, including apoptosis, cell signaling, and proliferation, its role in cisplatin-induced apoptosis in TNBC cells is not fully understood. The present study assessed calpain 1-dependent, cisplatin-induced apoptosis in TNBC cells. MATERIALS AND METHODS:MDA-MB231 cells were treated with different concentrations of cisplatin (0, 20, and 40 μM). The cisplatin deposit and its effect on endoplasmic reticulum and, subsequently, calcium release were detected using transmission electron microscopy and Von Koss staining, respectively. Calpain 1 messenger RNA, protein content, and apoptosis was measured using reverse transcriptase-polymerase chain reaction, Western blotting, and Hoechst stain, respectively. In addition, calpain modulation, by either activation or inhibition, and its effect on cisplatin-induced apoptosis were assessed. RESULTS:Our results showed that cisplatin induced endoplasmic reticulum stress, indicated by an increase in calcium staining and protein expression of glucose-regulated protein 78 and calmodulin, followed by cleavage of α-fodrin and caspase-12 and, eventually, apoptosis. Cyclopiazonic acid showed a similar effect and enhanced the sensitivity of these cells to cisplatin treatment. In contrast, calpain 1 inhibition by both specific small interfering RNA and exogenous inhibitor (calpeptin) attenuated cisplatin-induced apoptosis in these cells. CONCLUSION:Altogether, these findings suggest, for the first time, that calpain 1 activation by endoplasmic reticulum plays an essential role in sensitizing TNBC cells to cisplatin-induced apoptosis. This finding will allow exploration of new insights for the treatment of TNBC by overcoming its resistance to apoptosis. 10.1016/j.clbc.2016.12.001

Lamellar Inclusions within Hyperplastic Endoplasmic Reticulum in Benign Mesothelial Cells. Haefliger Simon,Jain Deepali,Menter Thomas,Vlajnic Tatjana,Savic Prince Spasenija,Hopfer Helmut,Mihatsch Michael J,Bubendorf Lukas Acta cytologica INTRODUCTION:In effusion cytology, mesothelial cells can occasionally present with striking intracytoplasmic accumulation of rod- and crystal-like cytoplasmic lamellar inclusions (LIs). Their nature and function are poorly understood, and their diagnostic relevance is unknown. OBJECTIVE:The aim of this study was to explore the nature of LIs in mesothelial cells and determine their prevalence and diagnostic utility in routine practice. MATERIAL AND METHOD:We reviewed a consecutive series of cytological specimens of reactive (n = 102) and malignant effusions (n = 90), respectively. Malignant effusions included malignant mesotheliomas (n = 63) and carcinomas (n = 27). LIs of one effusion were analyzed by electron microscopy (EM). RESULTS:LIs were found exclusively in benign mesothelial cells in 14% (14/102) of reactive and in 4% (1/27) of malignant effusions with carcinomatosis. They were absent in effusions of malignant mesothelioma. EM revealed mainly straight lamellar, less tubular, structures in cisternae of the hyperplasic rough endoplasmic reticulum (rER). CONCLUSION:Cytoplasmic LIs located within hyperplastic rER can be found in up to 14% of effusions restricted to benign mesothelial cells. They can be used as an indirect morphological clue favoring the diagnosis of benign effusion and helping the cytologist to differentiate between reactive and malignant mesothelial cells in daily practice. 10.1159/000508757

Diosgenyl Saponin Inducing Endoplasmic Reticulum Stress and Mitochondria-Mediated Apoptotic Pathways in Liver Cancer Cells. Meng Xin,Dong Honghong,Pan Yiwu,Ma Long,Liu Changxiao,Man Shuli,Gao Wenyuan Journal of agricultural and food chemistry Diosgenin and diosgenyl saponins as the major bioactive compounds isolated from dietary fenugreek seeds, yam roots, etc. possessed strong antitumor effects. To understand their detailed antitumor mechanisms, a fluorophore-appended derivative of diosgenin [Glc/CNHphth-diosgenin (GND)] was synthesized, starting from diosgenin and glucosamine hydrochloride in overall yields of 7-12% over 7-10 steps. Co-localization of GND with organelle-specific stains, transmission electron microscopy, and relative protein analyses demonstrated that GND crossed the plasma membrane through organic anion-transporting polypeptide 1B1 and distributed in the endoplasmic reticulum (ER), lysosome, and mitochondria. In this process, GND induced ER swelling, mitochondrial damage, and autophagosome and upregulating IRE-1α to induce autophagy and apoptosis. Furthermore, autophagy inhibitor chloroquine delayed the appearance of cleaved poly(ADP-ribose) polymerase and inhibited cleaved caspase 8, which indicated that GND induced autophagy to activate caspase-8-dependent apoptosis. These observations suggested that diosgenyl saponin was a potent anticancer agent that elicited ER stress and mitochondria-mediated apoptotic pathways in liver cancer. 10.1021/acs.jafc.9b05131