Background: Oridonin (ORI) could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood. Methods: Human prostate cancer (HPC) cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM). Chemical staining with acridine orange (AO), MDC or DAPI was used to detect acidic vesicular organelles (AVOs) and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS. Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC) cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs) were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA) reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%). Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy. Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand the anti-tumor mechanism of ORI as treatment for prostate cancer.

/pdf/oridonin-up-regulates-expression-of-p21-and-induces-1qlszj2oxc.pdf

Oridonin up-regulates expression of P21 and induces autophagy and apoptosis in human prostate cancer cells.

// Heather G Hambright 1 , Peng Meng 1, 6 , Addanki P Kumar 1, 2, 3, 4, 5 , Rita Ghosh 1, 2, 3, 4 1 Department of Urology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229, USA 2 Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229, USA 3 Department of Molecular Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229, USA 4 Cancer Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229, USA 5 South Texas Veterans Health Care System, San Antonio, Texas, 78229, USA 6 Life Sciences Division, Lawrence Berkley National Laboratory, Berkley, California, 94710, USA Correspondence to: Rita Ghosh, e-mail: ghoshr@uthscsaedu Keywords: Reactive oxygen species, oxidative stress, mTORC1, melanoma, Nexrutine R Received: December 22, 2014      Accepted: January 09, 2015      Published: January 29, 2015 ABSTRACT Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level Here we show that melanoma cells exhibit an oxidative stress phenotype compared with normal melanocytes, as evidenced by increased total cellular ROS, KEAP1/NRF2 pathway activity, protein damage, and elevated oxidized glutathione Our overall objective was to test whether augmenting this high oxidative stress level in melanoma cells would inhibit their dependence on oncogenic PI3K/AKT/mTOR-mediated survival We report that Nexrutine R augmented the constitutively elevated oxidative stress markers in melanoma cells, which was abrogated by N-acetyl cysteine (NAC) pre-treatment Nexrutine R disrupted growth homeostasis by inhibiting proliferation, survival, and colony formation in melanoma cells without affecting melanocyte cell viability Increased oxidative stress in melanoma cells inhibited PI3K/AKT/mTOR pathway through disruption of mTORC1 formation and phosphorylation of downstream targets p70S6K, 4EBP1 and rpS6 NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism Overall, our results illustrate the importance of disruption of the intrinsically high oxidative stress in melanoma cells to selectively inhibit their survival mediated by PI3K/AKT/mTOR

/pdf/inhibition-of-pi3k-akt-mtor-axis-disrupts-oxidative-stress-3pam0elmm3.pdf

Inhibition of PI3K/AKT/mTOR axis disrupts oxidative stress-mediated survival of melanoma cells.

Research studies focusing on various aspects of carcinogenesis in the background of fibrosis have advanced significantly in the recent past, allowing us to understand the mechanisms involved in malignant transformation of oral submucous fibrosis (OSF), the most prevalent potentially malignant oral disorder in South Asia. The role of areca nut as a carcinogen has been proven beyond doubt, with a large number of animal studies demonstrating its carcinogenicity, mutagenicity, and genotoxicity. Studies on the molecules implicated in cell cycle regulation, hypoxia, processes leading to DNA double-strand breaks, senescence, and many other pathways related to carcinogenesis have shown ample evidence for the malignant transformation in OSF induced by areca nut. More importantly, the understanding of the mechanisms of malignant transformation may lead to early diagnosis of oral squamous cell carcinoma arising in the background of OSF, which is now considered to constitute a clinicopathologically distinct disease, and the differences are believed to arise from the differential mechanisms of areca nut carcinogenesis. Therefore, the objective of this study is to review the literature on the various mechanisms leading to the malignant transformation of OSF.

Oral submucous fibrosis: review on mechanisms of malignant transformation.

Studies focusing on epidemiology, histopathology and molecular biology/pathology on various aspects of oral submucous fibrosis (OSF), especially in the last decade have helped to understand the pathogenesis to a larger extent. In addition research in some aspects of carcinogenesis in the background of fibrosis has also advanced significantly in the recent past allowing us to understand the mechanisms involved in malignant transformation of the most prevalent oral potentially malignant disorder in South Asia. It has been shown that pathogenesis of OSF is directly related to arecoline present in arecanut and most of the alterations in various pathways and molecules leading to accumulation of collagen are mediated as a result of arecoline. Reduction of Matrix metalloproteinases (MMPs) and increased secretion of Tissue inhibitors of matrix metalloproteinases (TIMPs) play the most significant role in collagen accumulation whilst fibrogenic cytokines, mainly TGF-β over expression leads to increased production of collagen. There are various other pathways/molecules contributing to the pathogenesis in varying capacities. Malignant transformation in OSF has also been studied by various groups in the recent past. Role of arecanut as a carcinogen is proven beyond doubt with a large number of animal studies demonstrating its carcinogenicity, mutagenicity and genotoxicity. Studies involved in many molecules implicated in cell cycle regulation, hypoxia, processes leading to DNA double strand breaks, senescence and many other pathways related to carcinogenesis have shown ample evidence for the arecanut induced malignant transformation in OSF. Some of the findings in these studies may be helpful in inventing new treatment strategies for a common disease without an effective treatment up to date. Further, the understanding of mechanisms of malignant transformation may lead to early diagnosis of oral squamous cell carcinoma (OSCC) arising in the background of OSF.

/pdf/oral-submucous-fibrosis-review-on-mechanisms-of-pathogenesis-4fxwe0rd8j.pdf

Oral Submucous Fibrosis: Review on Mechanisms of Pathogenesis and Malignant Transformation

Downregulated lncRNA-SNHG1 enhances autophagy and prevents cell death through the miR-221/222 /p27/mTOR pathway in Parkinson's disease.

Arecoline, the major alkaloid of areca nut, has been shown to cause strong genotoxicity and is considered a potential carcinogen. However, the detailed mechanism for arecoline-induced carcinogenesis remains obscure. In this study, we noticed that the levels of p21 and p27 increased in two oral squamous cell carcinoma cell lines with high confluence. Furthermore, when treated with arecoline, elevated levels of p21 and p27 could be downregulated through the reactive oxygen species/mTOR complex 1 (ROS/mTORC1) pathway. Although arecoline decreased the activity of mTORC1, the amounts of autophagosome-like vacuoles or type II LC3 remained unchanged, suggesting that the downregulation of p21 and p27 was independent of autophagy-mediated protein destruction. Arecoline also caused DNA damage through ROS, indicating that the reduced levels of p21 and p27 might facilitate G (1) /S transition of the cell cycle and subsequently lead to error-prone DNA replication. In conclusion, these data have provided a possible mechanism for arecoline-induced carcinogenesis in subcytolytic doses in vivo.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1349-7006.2012.02294.x

Arecoline downregulates levels of p21 and p27 through the reactive oxygen species/mTOR complex 1 pathway and may contribute to oral squamous cell carcinoma.

Areca nut has been proven to be correlated with various pathologic alterations in oral cavity. However, the mechanisms for such cytopathic effects are still elusive due mostly to the limitations of cell culture systems. Here we discovered that areca nut extract (ANE) induced production of autophagosome vacuoles in cells cultured with rich medium but induced pyknosis and ballooning, two morphological alterations frequently observed in betel quid chewers, in cells under a serum-free culture condition. Permeability of the serum-starved cells to propidium iodide (PI) confirmed ANE induced novel necrosis with pyknosis (pyknotic necrosis), providing a possible explanation for inflammatory infiltration in chewers’ mucosa. In these serum-starved cells, ANE strongly induced reactive oxygen species (ROS), which acted as a key switch for the initiation of pyknotic necrosis. Calcium flux was also involved in the morphological alterations. Besides, inhibition of GSK3β by SB216763 significantly exacerbated the pyknotic necrosis either induced by ANE or H2O2 in serum-starved cells, suggesting that GSK3β is a critical regulator for ANE/ROS-mediated pyknotic necrosis. Interestingly, LC3-II transition and PARP cleavage were still detected in the serum-starved cells after ANE treatment, suggesting concurrent activation of apoptotic and autophagic pathways. Finally, insulin could counteract the effect of ANE-induced pyknotic necrosis. Taken together, these data provide a platform for studying ANE-induced cytopathogenesis and the first clinical implication for several pathological alterations, such as ballooning and inflammatory infiltration, in betel quid chewers.

/pdf/areca-nut-extract-induces-pyknotic-necrosis-in-serum-starved-4o8n0ds45r.pdf

Areca nut extract induces pyknotic necrosis in serum-starved oral cells via increasing reactive oxygen species and inhibiting GSK3β: an implication for cytopathic effects in betel quid chewers.

Betel quid chewing is associated with various pathologic alterations in oral mucosa. However, the molecular mechanism behind so many contradictory alterations remains unclear. Here we aimed to build a model to facilitate the related studies in cultured cells. In our results, areca nut extract (ANE) was found to exert different effects in oral cells depending on the supplemented serum level. ANE strongly induced DNA damage, necrotic ballooning, and inflammatory cytokines under lower serum concentration while might convert to facilitate deregulated growth of serum-supplemented cells via modulating the activity/expression of factors such as E-cadherin and Snail. Despite ANE significantly activated NF-κB, a mediator critical for inflammation, inhibition of NF-κB did not prevent the activation of IL8 promoter. We further discovered Y705-dephosphorylated STAT3 might enhance IL8 transcription. Since necrosis and the inflammatory cytokines could cause massive inflammation, infiltration of interstitial fluid might potentiate cellular resistance against the acute cytotoxicity of ANE and further support the proliferation of transforming cells. Induction of VEGF and angiogenesis under lower serum condition also paved the way for cell growth and subsequent metastasis. Accordingly, we concluded that in correlation with serum infiltration ANE caused particular effects in oral cells and possibly the various clinicopathological alterations in vivo.

Areca nut extracts exert different effects in oral cancer cells depending on serum concentration: A clue to the various oral alterations in betel quid chewers.

Sheng-Ru Yang

Papers

Arecoline downregulates levels of p21 and p27 through the reactive oxygen species/mTOR complex 1 pathway and may contribute to oral squamous cell carcinoma.

Areca nut extract induces pyknotic necrosis in serum-starved oral cells via increasing reactive oxygen species and inhibiting GSK3β: an implication for cytopathic effects in betel quid chewers.

Areca nut extracts exert different effects in oral cancer cells depending on serum concentration: A clue to the various oral alterations in betel quid chewers.