Ceramides Increase Fatty Acid Utilization in Intestinal Progenitors to Enhance Stemness and Increase Tumor Risk.

BACKGROUND & AIMS: Cancers of the alimentary tract, including esophageal adenocarcinomas, colorectal cancers, and cancers of the gastric cardia, are common comorbidities of obesity. Prolonged, excessive delivery of macronutrients to the cells lining the gut can increase one's risk for these cancers by inducing imbalances in the rate of intestinal stem cell proliferation vs differentiation, which can produce polyps and other aberrant growths. We investigated whether ceramides, which are sphingolipids that serve as a signal of nutritional excess, alter stem cell behaviors to influence cancer risk. METHODS: We profiled sphingolipids and sphingolipid-synthesizing enzymes in human adenomas and tumors. Thereafter, we manipulated expression of sphingolipid-producing enzymes, including serine palmitoyltransferase (SPT), in intestinal progenitors of mice, cultured organoids, and Drosophila to discern whether sphingolipids altered stem cell proliferation and metabolism. RESULTS: SPT, which diverts dietary fatty acids and amino acids into the biosynthetic pathway that produces ceramides and other sphingolipids, is a critical modulator of intestinal stem cell homeostasis. SPT and other enzymes in the sphingolipid biosynthesis pathway are up-regulated in human intestinal adenomas. They produce ceramides, which serve as prostemness signals that stimulate peroxisome-proliferator activated receptor-α and induce fatty acid binding protein-1. These actions lead to increased lipid utilization and enhanced proliferation of intestinal progenitors. CONCLUSIONS: Ceramides serve as critical links between dietary macronutrients, epithelial regeneration, and cancer risk.

Anticancer Potential of Fruit Extracts from Vatica diospyroides Symington Type SS and Their Effect on Program Cell Death of Cervical Cancer Cell Lines.

Vatica diospyroides Symington is locally known as Chan-Ka-Pho in Thailand. Ancient people have used it as therapeutic plant for cardiac and blood tonic cure. The purpose of this study was to investigate the potential cytotoxicity and selectivity of the extracts from V. diospyroides type SS fruit on cervical cancer HeLa and SiHa cell lines and to examine its underlying mechanism of action. MTT assay revealed that the extracts showed inhibition of cell survival in a dose-dependent manner and exhibited highly cytotoxic activity against both HeLa and SiHa cells with IC50 value less than 20 µg/mL along with less toxicity against L929 cells. Acetone cotyledon extract (ACE) showed the best selectivity index value of 4.47 (HeLa) and 3.51 (SiHa). Distinctive morphological changes were observed in ACE-treated cervical cancer cells contributing to apoptosis action. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that ACE induced apoptosis in HeLa and SiHa cell lines in a dose-dependent manner. Treatment of ACE with half IC50 caused DNA fragmentation and also activated increasing of bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. The results suggest that ACE has potent and selective cytotoxic effect against cervical cancer cells and the potential to induce bax and caspase-8-dependent apoptosis. Hence, the ACE could be further exploited as a potential lead in cancer treatment.

Proteomic Analysis of Butyrate-Resistant Colorectal Cancer-Derived Exosomes Reveals Potential Resistance to Anti-Cancer Drugs.

BACKGROUND: Butyrate-resistant (BR) cells play an important role in acquiring chemoresistance in colorectal cancer (CRC). Our previous study demonstrated that BR CRC cells showed cross-resistance to chemotherapy drugs, including 5-fluorouracil and oxaliplatin, in both monolayer and spheroid cultures. The mechanisms underlying drug resistance were also elucidated. However, the link between parental (PT) and BR cells remains unclear. Extracellular vesicles (EVs) are key cell-cell communications that transport various molecules, including DNA, RNA, and proteins, between the donor and target cells. EVs contribute to drug resistance in cancers, such as melanoma and lung cancer. Recently, we focused on the correlation of proteomic profiles of EVs from different cell types. METHODS: In this study, we analyzed the proteomic profiles of EVs derived from PT and BR cells to investigate the mechanisms underlying the butyrate- and chemo-resistant phenotypes. EVs were isolated from PT and BR cells using ultracentrifugation. The characteristics of the EVs were evaluated using western blot and transmission electron microscopy. The EV proteomic data were further analyzed using liquid chromatography-mass spectrometry. RESULTS: We identified a unique protein expressed in BR cells related to the chemoresistant phenotype. Functional enrichment analysis showed that BR cells had higher protein catalytic activity, binding, and transcription activity. The STITCH database showed a greater correlation between protein-drug interactions in BR cells than in PT cells. Moreover, our findings support the hypothesis that EVs promote tumor progression and metastasis and affect the tumor microenvironment. CONCLUSIONS: Proteomic analysis of EVs from BR CRC cells reveals insights into drug resistance mechanisms, including protein-mediated carcinogenesis and reduced drug uptake, offering potential strategies to overcome resistance in clinical practice.

Validity evaluation of in-house preparation kit, vaginal pH paper test combined amine tube test, for the simple diagnosis of bacterial vaginosis.

OBJECTIVE: Determine the positive in-house preparation kit for suggested bacterial vaginosis (BV) for both elevated vaginal pH > 4.5 and positive amine test, as well as evaluate for validity of sensitivity, specificity, positive predictive value, and negative predictive value against Chandeying criteria for confirmed BV. MATERIAL AND METHOD: A cross-sectional study among the women who presented with an abnormal vaginal discharge (AVD) or asymptomatic annual cervical cytology screening was done. Each vaginal discharge was divided into two parts of investigation. The first part included the clinical criteria of confirmed BV, based on at least three out of five indicators, the vaginal pH > 4.5, homogeneous and thin discharge (milky discharge), positive sniff/amine test, clue cell > 20% of total vaginal epithelial cells, and scanty or absence lactobacilli. The second part included the in-house preparation kit of suggestive BV relied on elevated vaginal pH > 4.5 and positive amine tube test. RESULTS: Twenty-six women were enrolled. Of the complaint of AVD/asymptomatic had 2/10 of confirmed BV (12 cases), and 1/13 of confirmed non-BV (14 cases). The in-house preparation kit, compared with the clinical criteria, had sensitivity of 91%, specificity of 71%, positive predictive value of 73%, and negative predictive value of 90%. There were false negative of 1/12 cases (8.3%), and false positive of 4/14 cases (28.5%). CONCLUSION: The in-house preparation kit favorably compared with the clinical criteria and has the advantage of being simple, rapid, and easily performed in resource poor setting. Further development on sensitivity and specificity of the test is suggested.

Cytotoxic effect of metformin on butyrate-resistant PMF-K014 colorectal cancer spheroid cells.

Three-dimensional (3D) cell culture models are used in cancer research because they mimic physiological responses in vivo compared with two-dimensional (2D) culture systems. Recently, cross-resistance of butyrate-resistant (BR) cells and chemoresistance in colorectal cancer (CRC) cells have been reported; however, effective treatments for BR cells have not been identified. In this study, we investigated the cytotoxicity of metformin (MET), an anti-diabetic drug, on BR CRC cells in a 3D spheroid culture model. The results demonstrate that MET decreases spheroid size, migration, and spheroid viability, while it increases spheroid death. The molecular mechanism revealed that AMP-activated protein kinase (AMPK) and Akt serine/threonine kinase 1(Akt) were significantly upregulated, whereas the acetyl-CoA-carboxylase (ACC) and mammalian target of rapamycin (mTOR) were downregulated, which led to caspase activation and apoptosis. Our findings show the potential cytotoxicity of MET on CRC-BR cells. The combination of MET and conventional chemotherapeutic drugs should be addressed in further studies to reduce the side effects of standard chemotherapy for CRC.

Characterization of Butyrate-Resistant Colorectal Cancer Cell Lines and the Cytotoxicity of Anticancer Drugs against These Cells.

Colorectal cancer (CRC) is the third most common cancer worldwide. The gut microbiota plays a critical role in homeostasis and carcinogenesis. Butyrate, a short-chain fatty acid produced by the gut microbiota, plays a role in intestinal homeostasis and acts as an anticancer agent by inhibiting growth and inducing apoptosis. However, microbiota studies have revealed an abnormally high abundance of butyrate-producing bacteria in patients with CRC and indicated that it leads to chemoresistance. We characterized butyrate resistance in HCT-116 and PMF-K014 CRC cells after treatment with a maximum butyrate concentration of 3.2 mM. The 50% inhibitory concentration of butyrate was increased in butyrate-resistant (BR) cells compared with that in parental (PT) cells. The mechanism of butyrate resistance was initially investigated by determining the expression of butyrate influx- and drug efflux-related genes. We found the increased expression of influx- and efflux-related genes in BR cells compared with that in PT cells. Proteomic data showed both identical and different proteins in PT and BR cells. Further analysis revealed the crossresistance of HCT-116 cells to metformin and oxaliplatin and that of PMF-K014 cells to 5-fluorouracil. Our findings suggest that the acquisition of butyrate resistance induces the development of chemoresistance in CRC cells, which may play an important role in CRC development, treatment, and metastasis.

Barriers to cervical cancer screening and acceptability of HPV self-testing: a cross-sectional comparison between ethnic groups in Southern Thailand.

BACKGROUND: Cervical cancer rates are higher in low-resourced countries than high, partly due to lower rates of screening. Incidence in Thailand is nearly three times higher than in the USA (16.2 vs 6.5 age-standardised incidence), even with Thailand's universal health coverage, which includes screening, suggesting that alternative methods are needed to reduce the burden. We investigated barriers to screening, as well as acceptability of self-collection human papillomavirus (HPV) testing as a primary form of cervical cancer screening among Buddhist and Muslim communities in Southern Thailand. METHODS: 267 women from the Buddhist district of Ranot and Muslim district of Na Thawi, Songkhla were recruited to complete a survey assessing knowledge and risk factors of HPV and cervical cancer. Participants were offered an HPV self-collection test with a follow-up survey assessing acceptability. Samples were processed at Prince of Songkhla University and results were returned to participants. RESULTS: 267 women participated in the study (132 Buddhist, 135 Muslim), 264 (99%) self-collecting. 98% reported comfort and ease, and 70% preferred it to doctor-facilitated cytology. The main predictor of prior screening was religion (92% Buddhist vs 73% Muslim reporting prior Pap). After adjustment with multivariate logistic models, Muslim women had an OR of prior Pap of 0.30 compared with Buddhist (95% CI: 0.12 to 0.66). CONCLUSIONS: Self-collection HPV testing was highly acceptable across religious groups, suggesting that it could be beneficial for cervical cancer reduction in this region. Focus should be put into educating women from all backgrounds about the importance of screening to further improve screening rates among Thai women.

Molecular characterization of Hb H disease in southern Thailand.

Genotypes of 260 individuals with hemoglobin H (Hb H) disease originating from various provinces in southern Thailand were characterized by multiplex PCR (M-PCR) and reverse dot blot hybridization (RDB). M-PCR was used to amplify target fragments and then hybridized with allele-specific oligonucleotide (ASO) probes which were bound on a nylon membrane. A total of eight α-thalassemia (α-thal) mutations, which produced eight Hb H disease genotypes (α0-thal/α+-thal), were detected. The most common form of α0-thal was -SEA with a frequency of 99.23%. The other form (0.77%) of α0-thal mutation was a THAI deletion (-THAI). The deletional α+-thal mutations comprised 3.7 kb (-α3.7) and 4.2 kb (-α4.2) deletions which were found in 172 (66.15%) and 5 (1.92%) alleles, respectively. The incidence of non-deletional α+-thal in decreasing order was Hb Constant Spring (Hb CS, αCS) 28.85%, Hb Quong Sze (Hb QS, αQS) 1.54%, and Hb Paksé (Hb PS, αPS) 0.77%. The genotype characterization of Hb H disease and the development of the RDB technic for detection of α-thal mutations presented in this study enable the prenatal diagnosis of Hb Bart's hydrops fetalis syndrome.