Complementary Targeting of Rb Phosphorylation and Growth in Cervical Cancer Cell Cultures and a Xenograft Mouse Model by SHetA2 and Palbociclib

Abstract

Cervical cancer is caused by high-risk human papillomavirus (HPV) types and treated with conventional chemotherapy with surgery and/or radiation. HPV E6 and E7 proteins increase phosphorylation of retinoblastoma (Rb) by cyclin D1/cyclin dependent kinase (CDK)4/6 complexes. We hypothesized that cyclin D1 degradation by the SHetA2 drug in combination with palbociclib inhibition of CDK4/6 activity synergistically reduces phosphorylated Rb (phospho-Rb) and inhibits cervical cancer growth. The effects of these drugs, alone, and in combination, were evaluated in SiHa and CaSki HPV-positive and C33A HPV-negative cervical cancer cell lines using cell culture, western blots and ELISA, and in a SiHa xenograft model. Endpoints were compared by isobolograms, ANOVA, and Chi-Square. In all cell lines, combination indexes documented synergistic interaction of SHetA2 and palbociclib in association SHetA2 reduction of cyclin D1 and phospho-Rb, palbociclib reduction of phospho-Rb, and enhanced phospho-Rb reduction upon drug combination. Both drugs significantly reduced phospho-Rb and growth of SiHa xenograft tumors as single agents and acted additively when combined, with no evidence of toxicity. Dilated CD31-negative blood vessels adjacent to, or within, areas of necrosis and apoptosis were observed in all drug-treated tumors. These results justify development of the SHetA2 and palbociclib combination for targeting phospho-Rb in cervical cancer treatment.

Keywords: SHetA2; cervical cancer; cyclin D1; palbociblib; retinoblastoma.

Figure 1

Isobolograms of SHetA2 and palbociclib treatment on (A) C33A, (B) CaSki, and (C) SiHa cervical cancer cell lines.

Figure 2

Cyclin D1 levels in cultures treated with the indicated concentrations of SHetA2, palbociclib or the combination of these two drug for 24 h. (A) Representative western blots of C33A and CaSki cervical cancer cell lines are shown on top and graphs of cyclin D1 band intensities in replicate experiments are shown below. (B) Average of three independent cyclin D1 ELISA measurements in the indicated cell lines treated with the indicated drugs. ANOVA comparison of combination treatment with other treatment groups: ns = not significant, p value >0.05 and <0.01, * = p < 0.05, ** = p < 0.01, *** p < 0.001, **** p < 0.0001. The whole Western Blot see Figure S3.

Figure 3

Phospho-Rb levels in cultures treated with the indicated concentrations of SHetA2, palbociclib or the combination of these two drug for 24 h. Representative western blots of the indicated cervical cancer cell lines are shown on top. Graphs of phospho-Rb band intensities in replicate experiments are show below. ANOVA comparison of combination treatment with other treatment groups: ns = not significant, * = p < 0.05, ** = p < 0.01, *** p <0.001. The whole Western Blot see Figure S3.

Figure 4

Effect of control, SHetA2, palbociclib, and combination treatments on average tumor size (A) and body weight (B) in SiHa xenograft tumor bearing mice.

Figure 5

Effects of SHetA2 and palbociclib on SiHa xenograft tumor histology and target proteins. Representative sections of from the indicated treatment groups stained with H&E (Top row), trichrome stain (second row), CD31 (third row), cleaved caspase 3 (Active Casp 3), fourth row), cyclin D1 (fifth row), and phospho-Rb, sixth row). A = Abnormal dilated blood vessels, N = Normal blood vessel. The black line in each of the figures indicates 100 μm.

Figure 6

Stain scores of (A) cleaved caspase 3 and (B) phosphorylated pRb expression in SiHa xenograft tumors after the indicated treatments. Chi-Square Test for Trend * p = 0.03.