Fig 1.
Dose response curve of Apaziquone in oral cancer cells.
Alamar blue assay was performed to determine the LD50 of apaziquone in AMOS III, and SCC4 cells.
Fig 2.
Western Blot analysis of effect of apaziquone on apoptotic proteins in oral cancer cells.
Equal amounts of proteins in cell free extracts prepared from AMOS III and SCC4 cells treated with different doses of Apaziquone (250nM, 500nM and 1μM) and untreated as well as vehicle treated control cells were resolved by SDS-PAGE, transferred to PVP membranes, probed with specific antibodies and detected using ECL. Panels show- (A) AMOS III cells and (B) SCC4 cells. A dose dependent increase in cleaved PARP, cleaved Caspase 9 and cleaved Caspase 3 was observed in both oral cancer cell lines tested. β-actin was used as a loading control.
Fig 3.
Apaziquone treated AMOS III cells showed increased fragments of DNA as compared to the untreated vehicle control cells depicting increased apoptosis in drug treated cells.
Fig 4.
Annexin V assay and Cell Cycle analysis.
(A) Apaziquone treated AMOS III cells showed significant increase in early and late apoptosis (38%) as compared to the untreated (5%) and vehicle control cells (8%) by annexin V assay. (B) Apaziquone treated AMOS III cells show significantly higher number of cells in the Sub Go phase and G2M phases as compared to the untreated control AMOS III cells.
Fig 5.
In vivo effects of apaziquone treatment.
(A) No significant change in body weight was observed in apaziquone treated mice or the vehicle control mice during the course of treatment. (B)Effect of apaziquone treatment on tumor size in mice. Representative excised tumors depicting the difference in the size of tumors between apaziquone treated and the untreated control group mice. (C) Effect of Apaziquone treatment on tumor growth delay. Tumor xenografts developed in the flanks of NOD/SCID/CRL mice were administered with apaziquone (0.1 mg/kg body weight) intraperitoneal injections weekly for 6 weeks. Apaziquone treatment delayed the tumor growth significantly (p value < 0.001, paired two-tailed Student's t-test) in the drug treated group mice as compared to the mice in the untreated control or vehicle control groups. (D) Effect of Apaziquone treatment on kidney and liver of mice. Histology of liver and kidney tissues obtained at the conclusion of the in vivo study. Tissue sections were stained with hematoxylin and eosin (H&E). i: Section of liver after the treatment with vehicle control. ii: Section of liver after the treatment with apaziquone. iii: Section of the kidney after the treatment with vehicle control. iv: Section of kidney after the treatment with apaziquone. No oncocytic necrosis or fibrosis was observed in both kidney and liver after the treatment with apaziquone. v: IHC staining for Ki67 of untreated control oral tumor xenograft tissue section showing high nuclear Ki67 immunostaining and vi: apaziquone treated xenograft showing markedly reduced Ki67 staining in the apaziquone treated xenograft tumor tissue section (original magnification x 200).