Gonadal proliferation and reproductive cycle of the exotic sea squirt Cnemidocarpa amphora () (Pleurogona, Styelidae) sampled for the first time from the northern coast of Arabian Gulf in Saudi Arabia.

Specimens of Cnemidocarpa amphora were collected monthly from the Arabian Gulf from September 2017 to August 2018. Parts of their gonads were prepared for histological testing. The gonads' diameters varied by month. Each gonad contained many ovarian follicles with different morphologies and was surrounded by several testicular follicles. The ovarian and testicular follicles were separate, although the latter were always present near the former. Repeated measures ANOVA tests were conducted to investigate monthly changes in oocyte stages. In squirts measuring 12-13 cm in length, the gonads measured 30-50 mm from July to August; 20-25 mm from September to October; 15-20 mm from November to February; and 25-40 mm from March to June. Oogonia budded from the germinal epithelium with diameters of 20-30 µm. Previtellogenic oocytes measuring 70-120 µm were characterized by the deposition of small granules of protein around the nucleus, a continuous layer of follicular cuboidal epithelium, and scattered vacuoles in the ooplasm. The measurement of gonads and oocyte diameters were performed by image analysis (Image scope 2.3, Image Line, Inc.) and stage micrometer. The vitellogenic oocytes measured 130-220 µm and the follicular epithelium consisted of flattened and cuboidal layers. Beneath the vitelline membrane, scattered test cells appeared in the ooplasm and different granules of protein and MPS were deposited in the ooplasm. In the later phase, lipid droplets began to appear in the ooplasm. Yolk bodies formed after the impregnation of various granules together and the oocyte was ready to be shed. Before spawning, a yolk membrane appeared above the ooplasm. Post-vitellogenic oocytes, in which the homogeneity of ooplasm was restored, underwent gradual lysis and entered the atretic phase. Different stages of sperm development were present year-round in different follicles of the same squirt; hence, the testes were always mature.

Targeting the monocarboxylate transporter MCT2 and lactate dehydrogenase A LDHA in cancer cells with FX-11 and AR-C155858 inhibitors.

OBJECTIVE: In 1930, Otto Warburg reported that "aerobic glycolysis" is the intrinsic property of all tumor cells' fermentation of glucose to L-Lactate by lactate dehydrogenase A (LDHA) activity. This only produces per mole of glucose two moles of adenosine triphosphate (ATP), compared with 32 moles of ATP in a normal cell. Thus, tumor cells have to uptake 30 folds more glucose, the resulting accumulated lactate are then transported by a monocarboxylate transporter (MCT) with the participation of a CD147 molecule. Inhibition of MCT1 by RNA interference (RNAi) disrupted the unique metabolism of the tumor and caused tumor cell death. However, the effectiveness of the strategies depends on the targeted delivery of the therapeutics. MATERIALS AND METHODS: In this study, a synergistic approach was used to target LDHA and MCT1 with small molecule inhibitors FX11 and AR-C155858, respectively. Cell cytotoxicity assays (AlamarBlue assay), and Mitochondria Membrane Potential (JC-1) dye assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. To achieve this aim, the following objectives were proposed: the effect of metabolic inhibitors on tumor glycolytic metabolite environment, and the efficacy of metabolite inhibitors on human breast and colorectal cancer cells in vitro. Then, gene expression analysis was performed using Qiagen RT2 Profiler PCR array for apoptosis. All these assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. Normal human fibroblasts were used as control cells under normal and hypoxic culture conditions. RESULTS: In this study, the use of FX-11 inhibitors under normoxia or hypoxia in two or more cancer and normal cell lines has a direct effect on LDHA, whereby it inhibits its production, and this reduces the growth and cell proliferation of tumors. One of the more significant findings to emerge from this study is that using AR-C155858 inhibitor alone has increased the cell proliferation and showed no significant changes compared with the control. The other major finding was that combination of the two inhibitors, FX-11 and AR-C155858, under normoxia or hypoxia in two different cell lines MCF-7 and HCT-116 measured a decrease in the cells proliferative and red/green ratio. CONCLUSIONS: We successfully demonstrated that a combination of MCT1 inhibitor and LDHA inhibitor led to better outcomes. Indeed, this makes LDHA an ideal metabolic therapeutic target.

Immunotoxin-mediated targeting of claudin-4 inhibits the proliferation of cancer cells.

Immunotoxins are engineered chimeric proteins that consist of a fragment of a toxin fused to a modified antibody or growth factor capable of targeting specific cells. Furthermore, these proteins can be targeted to receptors that are commonly overexpressed on cancer cells. The majority of immunotoxins function by binding to cells, translocating into the cytosol and inhibiting protein synthesis. In this study, the expression of claudin­4 (CLDN4) in various cancer cells was analysed as a potential target for immunotoxins. To target CLDN4-expressing cancer cells, the c-terminal CLDN4­binding domain of Clostridium perfringens enterotoxin (CPE) was fused to the Pseudomonas aeruginosa exotoxin A (ETA) domain to create an immunotoxin (CPE­ETA'). Subsequently, the capacity of such an immunotoxin in suppressing the proliferation of CLDN4-positive cancer cells was investigated. We report that head and neck squamous carcinoma cells (HN5) have an elevated CLDN4 expression compared to the other cell lines tested. Our findings further demonstrate that CPE­ETA' is highly potent against MCF-7 breast [50% inhibitory concentration (IC50) 9.8 ng/ml] and HN5 head/neck (IC50 8.8 ng/ml) cancer cell lines, while it has no cytotoxic effects on HeLa cells (CLDN4­negative). The immunotoxin was subsequently expressed in the tumour colonising oncolytic strain, Clostridium ghonii. Most importantly, the strictly anaerobic Clostridium ghonii was able to overexpress and secrete a functional CPE­ETA' fusion protein. Our findings open the possibility of the targeted delivery of the immunotoxin locally to tumour sites at a high concentration using strictly anaerobic Clostridium ghonii for the treatment of CLDN4-positive cancer cells.