VJR‐TZ‐18: Novel Phosphatidylinositol‐3‐Kinase (PI3K) Delta Inhibitor Exerts Antitumor Activity via Induction of Autophagy and Apoptosis in Breast Cancer In Vitro and In Vivo
Authors: Lalita Guntuku , Kumar G Jagadeesh , Dinesh Thummuri , Rao Vaidya Jayathirtha , Naidu V G M 
Published: November 2023

Objective

Phosphatidylinositol‐3‐Kinase /mammalian target of rapamycin (PI3K/mTOR) pathway plays vital role in cancer by regulating critical cellular processes such as cell growth, metabolism and angiogenesis. In this study we aimed to screen novel triazine derivatives to identify potent anticancer drugs that act by inhibition of PI3K/mTOR pathway.

Methods

We screened novel triazine derivatives for their inhibitory activity on various isoforms of PI3K and cytotoxicity assays in a panel of human cancer cell lines. To unravel mechanism of action, we evaluated potent compound in assays such as acridine orange and ethidium bromide (AO/EB) staining, cell cycle analysis, Annexin V binding and JC‐1 assays in breast cancer cell lines. To determine the involvement of autophagy and apoptosis in its mode of action, Acidic vesicular organelle staining (AVO), LC3‐II immunofluorescence (IF) and western blotting for autophagy and apoptosis marker proteins were performed. In vivo acute toxicity, subacute toxicity, pharmacokinetic (PK) studies were performed in intraperitoneal (i.p) route in BALB/c mice to establish its safety. Anticancer efficacy was assessed by orthotropic breast cancer model established with MDA‐MB‐231/GFP cell line.

Results

PI3K isoform specific inhibitory assays revealed that among 32 novel triazine compounds, VJR‐TZ‐18 as a potent PI3K δ inhibitor with IC50 of 0.765 μM. This compound also displayed marked antitumor activity against breast cancer cell lines MDA‐MB‐231, MDA‐MB‐453 with IC50 values of 2.44, 2.37 μM respectively. Morphological studies AO/EB staining, Annexin V binding assays revealed the involvement of autophagy and apoptosis. Results of cell cycle analysis, JC‐1 assays suggest that VJR‐TZ‐18 arrest cell cycle at G0/G1, increase in sub G1 fraction and causes loss of mitochondrial membrane potential (MMP) in MDA‐MB‐231, MDA‐MB‐453 breast cancer cell lines. AVO staining, IF confirmed the induction of autophagy through increase in cells with positive AVO stain and marked elevation in LC3‐II puncta formation respectively. Further, western blotting analysis demonstrated the involvement of both autophagy and apoptosis by its elevation in their markers through activation of AMPK, inhibition of PI3K/mTOR. Maximum tolerated dose was found to be 400 mg/kg for VJR‐TZ‐18. In subacute toxicity testing, biochemical parameters and histopathology results did not show any toxicity at 22.5, 45 mg/kg doses. Pharmacokinetic studies at 22.5 mg/kg by i.p route have shown 2.6 hr t1/2, 1.4 μg/ml Cmax in mice. In vivo VJR‐TZ‐18 significantly decreased the tumor burden in tumor bearing mice when compared to control animals without major organ toxicities.

Conclusion

From the results it is clear that VJR‐TZ‐18 is a novel PI3K δ inhibitor displaying the potent anticancer activity in breast cancer in vitro and in vivo models. Thus VJR‐TZ‐18 may be developed as a novel PI3K δ inhibitor for the treatment in PI3K δ isoform over expressing cancers.

Support or Funding Information

Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India.