Deregulation of the CD44-NANOG-MDR1 associated chemoresistance pathways of breast cancer stem cells potentiates the anti-cancer effect of Kaempferol in synergism with Verapamil.

Chemoresistance is an imminent therapeutic challenge for breast cancer. Previous evidence suggests that breast cancer stem cells (BCSC) develop resistance through upregulation of stemness and chemo-evasion markers viz. SOX2, OCT4, NANOG, MDR1 and CD44, following anticancer chemotherapeutic treatments. Early studies suggest an inhibitory role of Kaempferol in BCSC propagation through downregulation of epithelial to mesenchymal transition. We hypothesized that the pathway involved in chemoresistance could be effectively addressed through Kaempferol (K), alone or in combination with Verapamil (V), which is an inhibitor of MDR1. We used K in combination with V, in multiple assays to determine if there was an inhibitory effect on BCSC. Both K and KV attenuated pH-dependent mammosphere formation in primary BCSC and MDA-MB-231 cells. RNA and protein (immunocytochemistry, western blot) expression of candidate markers viz. SOX2, OCT4, NANOG, MDR1 and CD44 were carried out in the presence or absence of candidate drugs in ex-vivo grown primary BCSC and MDA-MB-231 cell line. Immunoprecipitation assay, cell cycle analysis was carried out in MDA-MB-231. Our candidate drugs were not only anti-proliferative, but also downregulated candidate genes expression at RNA and protein level in both settings, with more robust efficacy in KV treatment than K; induced G2/M dependent cell cycle arrest, and interrupted physical association of CD44 with NANOG as well as MDR1 in MDA-MB-231. In primary tumor explant but not in adjacent normal tissue, our candidate drugs K and KV induced robust γH2AX expression. Thus, our candidate drugs are effective in attenuating BCSC survival.

Safety and Efficacy of Stosstherapy in Nutritional Rickets.

OBJECTIVE: Stosstherapy has been used since early 19th century for treating nutritional rickets. However, there are no clear cut guidelines for the biochemical monitoring of this treatment. Repeated blood tests at short intervals increase the cost of therapy and noncompliance. METHODS: A prospective study was conducted on 191 cases of nutritional rickets below 10 years of age to evaluate the effectivity of stosstherapy. All cases were treated with a single intramuscular injection of vitamin D (600.000 IU) along with oral calcium (50 mg/kg) and vitamin D (400 IU per day) until radiological resolution. Dietary modifications and adequate sunlight exposure were also recommended. RESULTS: The mean age of presentation was 2 years 9 months. Mean sunlight exposure was 17 minutes/week with 90% having low sunlight exposure (<30 minutes/week). Prolonged breast feeding (>6 months) was found in 93.7% of the cases. With treatment, the clinical features started resolving by 1 month with complete resolution of most of the features over a period of 1 year. By 6 months, all the study subjects had complete radiological resolution. Serum levels of calcium and alkaline phosphatase (ALP) were restored by 6 months in most cases while phosphate and vitamin D levels normalized by 6 weeks. CONCLUSION: Stosstherapy is a safe, cheap and effective method of treating nutritional rickets. Biochemical tests at initial presentation followed by vitamin D assay at 6 weeks and calcium, phosphate and ALP assays at 6 months is recommended in the monitoring of these patients. For regular monitoring, only ALP assay is recommended, provided one abstains from repeat injection of vitamin D based on high ALP levels.