The Effect of Novel 7-methyl-5-phenyl-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine Sulfonamide Derivatives on Apoptosis and Autophagy in DLD-1 and HT-29 Colon Cancer Cells.

Agnieszka Gornowicz,Anna Szymanowska,Anna Bielawska,Krzysztof Bielawski,Mariusz Mojzych

doi:10.3390/ijms21155221

Abstract

The discovery of cytotoxic drugs is focused on designing a compound structure that directly affects cancer cells without an impact on normal cells. The mechanism of anticancer activity is mainly related with activation of apoptosis. However, recent scientific reports show that autophagy also plays a crucial role in cancer cell progression. Thus, the objective of this study was to synthesize 7-methyl-5-phenyl-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine utilizing nucleophilic substitution reaction at the position N1. The biological activity of tested compounds was assessed in DLD-1 and HT-29 cell lines. The induction of apoptosis was confirmed by Annexin V binding assay and acridine orange/ethidium bromide staining. The loss of mitochondrial membrane potential and caspase-8 activity was estimated using cytometer flow analysis. The concentration of p53, LC3A, LC3B and beclin-1 was measured using the ELISA technique. Our study revealed that anticancer activity of 7-methyl-5-phenyl-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine derivatives is related with initiation of apoptosis occur on the intrinsic pathway with mitochondrial membrane decrease and extrinsic with increase of activity of caspase-8. Moreover, a decrease in beclin-1, LC3A, and LC3B were observed in two cell lines after treatment with novel compounds. This study showed that novel 7-methyl-5-phenyl-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine derivatives might be a potential strategy in colon cancer treatment.

Highlights

Colorectal cancer (CRC) represents a major health problem and ranks as the second most lethal malignancy worldwide
The apoptosis is triggered by the activation of caspase-8 and decrease of mitochondrial membrane potential, but the most significant effect of treatment was observed in DLD-1 cells, which are more sensitive to chemotherapy than HT-29 cells [28]
Autophagy may be responsible for resistance of cancer cells to many chemotherapeutic agents [31], and it can be seen as an attractive target for the treatment of patients with colorectal cancer, where almost 20% of them develop metastases

Summary

Introduction

Colorectal cancer (CRC) represents a major health problem and ranks as the second most lethal malignancy worldwide. Chemotherapy, and targeted therapy are still the most common methods of treatment of patients with CRC. Chemotherapy is based on single agents such as fluoropyrimidine (5-FU) and multiple agent therapy, which includes oxaliplatin, capecitabine, and irinotecan. Some adverse effects and limitations such as increased toxicity, resistance and low selectivity towards cancer cells give the basis to look for novel approaches and new chemotherapeutic agents. Other therapeutic strategies involve antibodies (cetuximab, panitumumab, bevacizumab) against molecular targets such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) [1]. Research on the development of a cytotoxic drug is aimed at designing a compound structure, whose action is directed at cancer cells while not affecting normal cells

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