Heterogeneous Differentiation of Highly Proliferative Embryonal Carcinoma PCC4 Cells Induced by Curcumin: An In Vitro Study.

Curcumin, the yellow pigment derived from turmeric rhizomes, exhibits antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. We have previously reported in a study that curcumin could induce differentiation in embryonal carcinoma cell (EC). EC cells are the primary constituents of teratocarcinoma tumors, and hence differentiating them to a non-proliferative cell type may be useful in anticancer therapies. Here, we conducted a detailed study using various molecular approaches to characterize this differentiation at the cellular and molecular levels. The cells were treated with 20 µM curcumin, which was the optimal concentration to produce the highest amount of differentiated cells. Changes in protein and RNA expression, membrane dynamics, and migration of these cells after treatment with curcumin were then studied in a time-dependent manner. The differentiated cells were morphologically distinct from the precursor cells, and gene expression profiles were altered in curcumin-treated cells. Curcumin promoted cell motility and cell adhesion. Curcumin also induced changes in membrane fluidity and the lateral mobility of lipids in the plasma membrane. The findings of this study suggest that curcumin might have therapeutic potential in differentiation therapy for the treatment of teratocarcinomas or germ cell tumors (GCTs) such as testicular and ovarian GCTs.

Etiological profile of diarrhea in solid organ transplant recipients at a tertiary care center in Southern India.

BACKGROUND: Diarrhea is one of the common gastrointestinal (GI) adverse events after solid organ transplantation. Diarrhea may be caused by infectious or non-infectious etiology. The infectious etiology of diarrhea varies according to the location and duration of diarrhea. Non-infectious etiologies include drugs, inflammatory bowel disease, neoplasia. The objective of this study was to evaluate the etiological profile of diarrhea in solid organ transplant recipients presenting to a tertiary care center in Southern India. METHODS: This was a retrospective analysis of prospectively collected data of all solid organ transplantation recipients referred to the Department of Medical Gastroenterology for evaluation of diarrhea from April 2012 till May 2014. All patients had stool evaluated by wet mount examination, modified acid fast (AFB) stain, trichrome stain, culture, and Clostridium difficile toxin assay. EDTA plasma was collected for quantitative Cytomegalovirus (CMV) detection by real-time PCR. If the diarrhea was acute (<2 wk), and no etiological agent was identified, empirical antibiotic therapy was instituted and followed up. If persistent or chronic diarrhea (>2-4 wk), endoscopic evaluation (upper GI endoscopy and/or colonoscopy with biopsies), depending on the clinical type of diarrhea was done. If no specific etiological diagnosis was established after endoscopic evaluation, breath test for SIBO and celiac serology were done. If no specific etiology was identified after the above investigations, dose of immunosuppressive drugs was reduced. If diarrhea responded to dose reduction, it was considered to be drug related. RESULTS: Fifty-eight episodes of diarrhea occurred in 55 solid organ transplant recipients during the study period. Renal transplant recipients constituted the majority (70%). Most (79%) of patients included in the study had their transplant > 6 mo ago. Infective diarrhea was the etiology in 46%, drug-related diarrhea in 29.3%. No specific etiology was identified in 22.4% of patients. Parasites accounted for 69% of all infective diarrhea. Stool evaluation was the main investigation in establishing diagnosis in acute diarrhea. Endoscopic evaluation was required in two thirds of patients to establish diagnosis in chronic diarrhea. CONCLUSION: GI infections and drug-related diarrhea were the common causes of diarrhea in solid organ transplant recipients. Parasites were the most common infectious etiology of diarrhea. Step-wise evaluation was able to identify the etiology in ~ 77% of patients. Overall, 98% of diarrheal episodes resolved.

Atypical heat shock response and acquisition of thermotolerance in P388D1 cells.

Reports of atypical heat shock response in some tumour cell lines emphasize the possibilities of alternate stress response mechanisms. We demonstrate here that P388D1, a mouse macrophage tumour cell line, failed to induce heat shock proteins (HSPs) in response to either heat stress (42 °C, 1h) or to heavy metal stress induced by arsenic trioxide (5-20 µM). Heat shock transcriptional factor 1 (HSF1) that mediates transcriptional up regulation of HSPs during stress was found to be deficient in transactivation despite its binding to the promoter region of HSP genes. Interestingly, cells exhibited thermotolerance in the absence of induced HSPs. However, the tolerance was abrogated in cells treated with cycloheximide (250 ng/ml) suggested that thermotolerance was dependent on de novo protein synthesis.

Cullin4B/E3-ubiquitin ligase negatively regulates beta-catenin.

Beta-catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, beta-catenin is targeted to ubiquitin-proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in beta-catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased beta-catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and beta-catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of beta-catenin levels.

Heat shock transcription factors regulate heat induced cell death in a rat histiocytoma.

Heat shock response is associated with the synthesis of heat shock proteins (Hsps) which is strictly regulated by different members of heat shock transcription factors (HSFs). We previously reported that a rat histiocytoma, BC-8 failed to synthesize Hsps when subjected to typical heat shock conditions (42 degrees C, 60 min). The lack of Hsp synthesis in these cells was due to a failure in HSF1 DNA binding activity. In the present study we report that BC-8 tumor cells when subjected to heat shock at higher temperature (43 degrees C, 60 min) or incubation for longer time at 42 degrees C, exhibited necrosis characteristics; however,under mild heat shock (42 degrees C, 30 min) conditions cells showed activation of autophagy. Mild heat shock treatment induced proteolysis of HSF1, and under similar conditions we observed an increase in HSF2 expression followed by its enhanced DNA binding activity. Inhibiting HSF1 proteolysis by reversible proteasome inhibition failed to inhibit heat shock induced autophagy. Compromising HSF2 expression but not HSF1 resulted in the inhibition of autophagy, suggesting HSF2 dependent activation of autophagy. We are reporting for the first time that HSF2 is heat inducible and functions in heat shock induced autophagic cell death in BC-8 tumor cells.

Effect of C-terminal deletion of P53 on heat induced CD95 expression and apoptosis in a rat histiocytoma.

Tumor suppressor gene product p53 in its wild-type conformation, is an effector of apoptosis. A rat histiocytic tumor, AK-5 which has a rearranged and mutated p53 gene undergoes apoptosis upon heat shock through surface expression of CD95 receptor. DNA sequence analysis of p53 gene from tumor cells revealed a deletion of 'C' at nucleotide position 942 and an addition of 'A' at position 1055. Deletion of one nucleotide caused premature termination of p53 protein which resulted in shorter p53 protein with an altered sequence from amino acids 315 to 341. Altered p53 was unable to protect BC-8, a single cell clone of AK-5 cells from apoptosis upon heat shock. BC-8 cells transfected with a wild-type p53gene (3B4 cells) were resistant to heat induced apoptosis and did not show the expression CD95 death receptor. Inhibition of p53 expression by using antisense oligo induced apoptosis upon heat shock in 3B4 cells. Similarly, inhibition of CD95 expression by antisense oligo inhibited heat induced apoptosis in BC-8 cells. In addition, cell cycle regulatory molecules, cdc2 and cdk2 are differentially regulated in a non-cell cycle dependent manner in these tumor cells. These results, in view of lack of heat shock response in BC-8 cells suggest a complex interaction between p53, CD95 and hsp70 which determines the fate of the cell. In the absence of functional p53, CD95 appears to be an effector of apoptosis in BC-8 cells.

Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase.

Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5'extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/modification of nuclear proteins.

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma.

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.

Heat shock induces chromosomal instability in near-tetraploid embryonal carcinoma cells.

Embryonal carcinoma cells, the cancer stem cell of teratocarcinoma, provide a good model system to study various aspects of embryonic and cancer development. Abnormal karyotype not only affects the normal development and differentiation, but also boosts tumor development. As evidences suggesting the role of karyotypic abnormalities (e.g., aneuploidy and near-tetraploidy) and stem cell in tumor development are increasing, it becomes pertinent to study karyotypically abnormal embryonal carcinoma cells. We isolated a subclone, P19RG01, from long-term culture of P19 embryonal carcinoma cell line. This subclone is near-tetraploid and expresses markers of embryonal carcinoma cells. Here we show that sub-lethal heat shock induces centrosome amplification, multipolar mitosis and chromosome missegregation thereby inducing chromosomal instability by several fold. The formation of supernumerary centrosome is accompanied by a long G(2) arrest. These events lead to the generation of heterogeneous population of cells in culture. Our observations support the previous observations that selective forces (both intrinsic as well as extrinsic) play an important role in tumor progression. Moreover, we provide direct evidences which suggest chromosomal instability in cancer stem cells, using P19RG01 embryonal carcinoma cells as a model system. We propose that a minor population of chromosomally unstable cancer stem cells exists in a tumor or is generated (transient existence) in the presence of selective forces. These chromosomally unstable cancer stem cells generate a heterogeneous cancer stem cell population, which undergoes Darwinian selection, leading to selection of more malignant cancer stem cells and thereby tumor progression.

Cell cycle regulation by the pro-apoptotic gene Scotin.

Scotin is a pro-apoptotic mammalian gene, which is induced upon DNA damage or cellular stress in a p53-dependent manner. In this report, we have used Drosophila as a model system to obtain a preliminary insight into the molecular mechanism of Scotin function, which was further validated using the mammalian system. Targeted expression of Scotin in developing Drosophila induced apoptosis and developmental defects in wings and eyes. Co-expression of Scotin with the anti-apoptotic protein p35, while inhibited the apoptosis in both dividing and non-dividing cells, rescued adult wing or eye phenotypes only when Scotin was expressed in non-dividing cells. This suggests that mechanisms of Scotin-induced apoptosis in dividing and non-dividing cells may vary. Suppressor-enhancer screen using cell cycle regulators suggested that Scotin may mediate cell cycle arrest at both G(1)/S and G(2)/M phases. Overexpression of Scotin in mammalian cells resulted in mitotic arrest and subsequently apoptosis. Furthermore, a larger proportion of cells overexpressing Scotin showed sequestration of Cyclin B1 in the cytoplasm. These results suggest that one of the ways by which Scotin induces apoptosis is by causing cell cycle arrest.

Alpha-fetoprotein as a tumor marker in hepatocellular carcinoma: investigations in south Indian subjects with hepatotropic virus and aflatoxin etiologies.

OBJECTIVES: The prevalence of hepatitis B virus (HBV) is reportedly the main cause of hepatocellular carcinoma (HCC) in India, where hepatitis C virus (HCV)-associated HCC is believed to be relatively less prevalent. We verified the usefulness of alpha-fetoprotein (AFP) as a tumor marker and analyzed the influence of viral etiology on AFP levels in HCC. METHODS: Of a total of 1012 cases with liver disease, 202 were investigated for the presence of AFP (142 HCC cases, 30 cirrhosis cases, and 30 chronic liver disease (CLD) cases). In addition, serum samples from 30 healthy patients, 30 hepatitis B surface antigen (HBsAg) carriers, and 30 acute viral hepatitis cases were included as controls. AFP was quantitatively determined using a commercial ELISA (Quorum Diagnostics, Canada). Out of the 142 HCC cases screened for AFP, aflatoxin B1 (AFB1) detection was carried out in 38 HCC cases using an in-house immunoperoxidase test. RESULTS: In HBV and HCV co-infected HCC cases, the AFP positivity was 85.7%. In HBV alone-associated HCC, the positivity was 62.9%, and 54.5% of AFB1 positive HCC cases showed AFP positivity. In HBV and HCV negative HCC cases, the positivity was 20.5%, and in HCV-associated HCC it was 17.6%. The HBV/HCV co-infected group and HBV alone positive HCC cases had significantly elevated levels of AFP. When AFP positivity was analyzed based on the marker profile of HBV, 89.7% of AFP positive cases were HBV-DNA positive. CONCLUSIONS: The overall positivity pattern of AFP in HCC does indicate that higher levels of AFP are observed with hepatitis virus positivity, especially with HBV. Further studies must be carried out to correlate the serum levels of AFP with the size, number, and degree of differentiation of HCC nodules.

Activation of stress response by ionomycin in rat hepatoma cells.

All living systems respond to a variety of stress conditions by inducing the synthesis of stress or heat shock proteins (HSPs), which transiently protect cells. HSP synthesis was preceded by an increase in intracellular free calcium concentration [(Ca(2+))i]. In this study, we show that Ca(2+) ionophore, ionomycin, induced an immediate increase in intracellular free Ca(2+) and examined how this increase affects heat shock response in rat hepatoma cell line H4II-E-C3. Results indicate that incubating H4II-E-C3 cells with 0.3 microM ionomycin at 37 degrees C for 15 min results in the induction of HSP 70 in both Ca(2+)-containing and Ca(2+)-free medium. Associated with this increase in free Ca(2+) is an in vivo change in membrane organization and activation of signaling molecules like ERKS and SAPKs/JNK. In Ca(2+) containing medium HSP 70 induction mediated by HSF-HSE interaction was faster upon ionomycin treatment as compared to heat shock. Our results show that ionomycin, at sub lethal concentration, increases intracellular free Ca(2+) concentration, activates SAPK/JNK and HSF-HSE interaction, and induces HSP 70 synthesis.