Boyun Kim

Curcumin induces ER stress-mediated apoptosis through selective generation of reactive oxygen species in cervical cancer cells

Prolonged accumulation of misfolded or unfolded proteins caused by cellular stress, including oxidative stress, induces endoplasmic reticulum stress, which then activates an unfolded protein response (UPR). ER stress is usually maintained at higher levels in cancer cells as compared to normal cells due to altered metabolism in cancer. Here, we investigated whether curcumin is ER stress‐mediated apoptosis in cervical cancer cells, and ROS increased by curcumin are involved in the process as an upstream contributor. Curcumin inhibited proliferation of cervical cancer cells (C33A, CaSki, HeLa, and ME180) and induced apoptotic cell death. Curcumin activated ER‐resident UPR sensors, such as PERK, IRE‐1α, and ATF6, and their downstream‐signaling proteins in cervical cancer cells, but not in normal epithelial cells and peripheral blood mononuclear cells (PBMCs). CHOP, a key factor involved in ER stress‐mediated apoptosis, was also activated by curcumin. CHOP decreased the ratio of anti‐apoptotic protein Bcl‐2 to pro‐apoptotic protein Bax expression, and subsequently increased the apoptotic population of cervical cancer cells. Furthermore, curcumin elevated levels of intracellular reactive oxygen species (ROS) in cervical cancer cells, but not in normal epithelial cells. Scavenging ROS resulted in inhibition of ER stress and partially restored cell viability in curcumin‐treated cancer cells. Collectively, these observations show that curcumin promotes ER stress‐mediated apoptosis in cervical cancer cells through increase of cell type‐specific ROS generation. Therefore, modulation of these differential responses to curcumin between normal and cervical cancer cells could be an effective therapeutic strategy without adverse effects on normal cells.

Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells

Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells.

Tumor evolution and intratumor heterogeneity of an epithelial ovarian cancer investigated using next-generation sequencing

BackgroundThe extent to which metastatic tumors further evolve by accumulating additional mutations is unclear and has yet to be addressed extensively using next-generation sequencing of high-grade serous ovarian cancer.MethodsEleven spatially separated tumor samples from the primary tumor and associated metastatic sites and two normal samples were obtained from a Stage IIIC ovarian cancer patient during cytoreductive surgery prior to chemotherapy. Whole exome sequencing and copy number analysis were performed. Omental exomes were sequenced with a high depth of coverage to thoroughly explore the variants in metastatic lesions. Somatic mutations were further validated by ultra-deep targeted sequencing to sort out false positives and false negatives. Based on the somatic mutations and copy number variation profiles, a phylogenetic tree was generated to explore the evolutionary relationship among tumor samples.ResultsOnly 6% of the somatic mutations were present in every sample of a given case with TP53 as the only known mutant gene consistently present in all samples. Two non-spatial clusters of primary tumors (cluster P1 and P2), and a cluster of metastatic regions (cluster M) were identified. The patterns of mutations indicate that cluster P1 and P2 diverged in the early phase of tumorigenesis, and that metastatic cluster M originated from the common ancestral clone of cluster P1 with few somatic mutations and copy number variations.ConclusionsAlthough a high level of intratumor heterogeneity was evident in high-grade serous ovarian cancer, our results suggest that transcoelomic metastasis arises with little accumulation of somatic mutations and copy number alterations in this patient.

Original ArticlesCurcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells

Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells.

Ascites modulates cancer cell behavior, contributing to tumor heterogeneity in ovarian cancer.

Malignant ascites constitute a unique tumor microenvironment providing a physical structure for the accumulation of cellular and acellular components. Ascites is initiated and maintained by physical and biological factors resulting from underlying disease and forms an ecosystem that contributes to disease progression. It has been demonstrated that the cellular contents and the molecular signatures of ascites change continuously during the course of a disease. Over the past decade, increasing attention has been given to the characterization of components of ascites and their role in the progression of ovarian cancer, the most malignant gynecologic cancer in women. This review will discuss the role of ascites in disease progression, in terms of modulating cancer cell behavior and contributing to tumor heterogeneity.

ROS Accumulation by PEITC Selectively Kills Ovarian Cancer Cells via UPR-Mediated Apoptosis.

Unfolded protein response (UPR) is crucial for both survival and death of mammalian cells, which is regulated by reactive oxygen species (ROS) and nutrient depletion. In this study, we demonstrated the effect of ROS-accumulation, induced by β-phenethyl isothiocyanate (PEITC), on UPR-mediated apoptosis in ovarian cancer cells. We used ovarian cancer cell lines, PA-1 and SKOV-3, with different p53 status (wild- and null-type, respectively). PEITC caused increased ROS-accumulation and inhibited proliferation selectively in ovarian cancer cells, and glutathione (GSH) depletion in SKOV-3. However, PEITC did not cause any effect in normal ovarian epithelial cells and peripheral blood mononuclear cells. After 48 h of PEITC treatment (5 μM), apoptotic cell death was shown to increase significantly in the ovarian cancer cells and not in the normal cells. The key regulator of UPR-mediated apoptosis, CHOP/GADD153 and endoplasmic reticulum resident chaperone BiP/GRP78 were parallely up-regulated with activation of two major sensors of the UPR [PERK and ATF-6 in PA-1; PERK, and IRE1α in SKOV-3) in response to ROS accumulation induced by PEITC (5 μM). ROS scavenger, N-acetyl-L-cysteine (NAC), attenuated the effect of PEITC on UPR signatures (P-PERK, IRE1α, CHOP/GADD153, and BiP/GRP78), suggesting the involvement of ROS in UPR-mediated apoptosis. Altogether, PEITC induces UPR-mediated apoptosis in ovarian cancer cells via accumulation of ROS in a cancer-specific manner.

Autophagy and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 alpha kinase (eIF2α) pathway protect ovarian cancer cells from metformin-induced apoptosis.

Metformin, an oral biguanide for the treatment of type II diabetes, has been shown to have anticancer effects in ovarian cancer. Energy starvation induced by metformin causes endoplasmic reticulum stress‐mediated unfolded protein response (UPR) and autophagy. UPR and autophagy act as a survival or death mechanism in cells. In this study, we observed that metformin‐induced apoptosis was relieved by autophagy and the PERK/eIF2α pathway in ovarian cancer cells, but not in peripheral blood mononuclear cells (PBMC) or ‘normal’ ovarian surface epithelial cells (OSE). Increased PARP cleavage and increased LC3B‐II with ATG5‐ATG12 complex suggested the induction of apoptosis and autophagy, respectively, in metformin‐treated ovarian cancer cells. Accumulation of acidic vacuoles in the cytoplasm and downregulation of p62 further supported late‐stage autophagy. Interestingly, metformin induced interdependent activation between autophagy and the UPR, especially the PERK/eIF2α pathway. Inhibition of autophagy‐induced PERK inhibition, and vice versa, were demonstrated using small molecular inhibitors (PERK inhibitor I, GSK2606414; autophagy inhibitor, 3‐MA, and BafA1). Moreover, autophagy and PERK activation protected ovarian cancer cells against metformin‐induced apoptosis. Metformin treatment in the presence of inhibitors of PERK and autophagy, however, had no cytotoxic effects on OSE or PBMC. In conclusion, these results suggest that inhibition of autophagy and PERK can enhance the selective anticancer effects of metformin on ovarian cancer cells.

Mitochondrial dynamics altered by oxidative stress in cancer

Abstract Mitochondria as crucial organelles regulate cellular energy generation, calcium and redox homeostasis, and apoptosis. To perform the cellular functions effectively, mitochondria continuously change their structure and morphology through protein machineries controlling fission and fusion process (mitochondrial dynamics). Traditionally, many researches had focused on the interaction of mitochondrial dynamics and apoptosis. However, recent studies are reporting the alteration of mitochondrial dynamics in human diseases including many types of cancers. Considering that cancers maintain a high level of reactive oxygen species (ROS), mitochondrial dynamics can be influenced by oxidative stress. In this review, we will discuss the alteration of mitochondrial dynamics by ROS and its effect on metastasis and chemoresistance in cancers.

Malignant ascites enhances migratory and invasive properties of ovarian cancer cells with membrane bound IL-6R in vitro

Transcoelomic route is the most common and the earliest route of metastasis, causing the ascites formation in advanced epithelial ovarian cancer (EOC). We demonstrated that interleukin 6 (IL-6) is enriched in the malignant ascites from patients with ovarian cancer, which enhanced invasive properties of EOC cells. Interestingly, the expression of IL-6R on cell membrane of EOC cells correlated with ascites-induced invasion. Selective knockdown of IL-6R or inhibition with IL-6 neutralizing antibody, suppressed the stimulatory effects of ascites on EOC invasion. Moreover, the ascites treatment induced the phosphorylation of JAK2-STAT3 and use of selective inhibitors of JAK2 and STAT3, blocked the expression of epithelial-mesenchymal transition related proteins in parallel with the suppression of EOC invasion. Thus, IL-6/IL-6R mediated JAK2-STAT3 signaling pathway could be a promising therapeutic target for anticancer therapy in ovarian cancer patients with ascites.

Adipose Stromal Cells from Visceral and Subcutaneous Fat Facilitate Migration of Ovarian Cancer Cells via IL-6/JAK2/STAT3 Pathway.

Purpose Adipose stromal cells (ASCs) play an important regulatory role in cancer progression and metastasis by regulating systemic inflammation and tissue metabolism. This study examined whether visceral and subcutaneous ASCs (V- and S-ASCs) facilitate the growth and migration of ovarian cancer cells. Materials and Methods CD45– and CD31– double-negative ASCs were isolated from the subcutaneous and visceral fat using magnetic-activated cell sorting. Ovarian cancer cells were cultured in conditioned media (CM) obtained from ASCs to determine the cancer-promoting effects of ASCs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, Boyden chamber assay, and western blotting were performed to determine the proliferative activity, migration ability, and activation of the JAK2/STAT3 pathway, respectively. Results CM from ASCs enhanced the migration of the ovarian cancer line, SKOV3, via activation of the JAK2/STAT3 signaling pathway. Interestingly, in response to ASC-CM, the ascites cells derived from an ovarian cancer patient showed an increase in growth and migration. The migration of ovarian cancer cells was suppressed by blocking the activation of JAK2 and STAT3 using a neutralizing antibody against interleukin 6, small molecular inhibitors (e.g., WP1066 and TG101348), and silencing of STAT3 using siRNA. Anatomical differences between S- and V-ASCs did not affect the growth and migration of the ovarian cancer cell line and ascites cells from the ovarian cancer patients. Conclusion ASCs may regulate the progression of ovarian cancer, and possibly provide a potential target for anticancer therapy.