Abstract
Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC50 value was 1.42±0.11 µM for NMK-TD-100 for 48 h treatment. In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC50 value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.
Highlights
Worldwide, cervical cancer is considered to be the second most common form of cancer as far as mortality and incidence are concerned and India contributes to about 20–30% of the global burden [1]
The anti-proliferative effect of some synthetic 5-(3-indolyl)-2substituted-1,3,4-thiadiazoles was screened using human cervical epithelial carcinoma (HeLa) by MTT assay and it was found that NMK-TD-100 (Fig. 1A for Structure) was the most effective one among all the compounds in inhibiting the proliferation of the HeLa cells
The cytotoxicity of NMK-TD-100 in normal peripheral blood mononuclear cells (PBMC) was examined and we found that it was less toxic towards PBMC
Summary
Cervical cancer is considered to be the second most common form of cancer as far as mortality and incidence are concerned and India contributes to about 20–30% of the global burden [1]. Cervical cancer is the most common malignancy among Indian women. Over a span of 25-year, the number of cases of cervical cancer has steadily increased in India, with over 80% of cases occurring amongst rural women. The treatment of cervical cancer varies with the stages of development of the cancer. Stage cancers can be eradicated by surgery and radiation therapy. Advanced stage tumors are treated with radiation therapy and cisplatin-based chemotherapy. In 2006, the US Food and Drug Administration approved the use of a combination of two chemotherapy drugs, hycamtin and cisplatin for women with late-stage cervical cancer treatment [3]. There is always a quest for new chemotherapeutic agents which will be effective in killing the cervical cancer cells with minimal toxicity to the subject
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