A triphenylethylene nonsteroidal SERM attenuates cervical cancer growth

Abstract

Selective estrogen receptor modulator drug molecules of triphenylethylene family have gained considerable attention as anti-cancer agents. Despite recent advances in screening and development of HPV vaccines, cervical cancer remains one of the deadliest malignancies as advanced stage metastatic disease is mostly untreatable, thus warrants newer therapeutic strategies. Ormeloxifene (ORM) is a well-known SERM of triphenylethylene family that has been approved for human use, thus represents an ideal molecule for repurposing. In this study, we for the first time have demonstrated the anti-cancerous properties of ormeloxifene in cervical cancer. Ormeloxifene efficiently attenuated tumorigenic and metastatic properties of cervical cancer cells via arresting cell cycle at G1-S transition, inducing apoptosis, decreasing PI3K and Akt phosphorylation, mitochondrial membrane potential, and modulating G1-S transition related proteins (p21, cyclin E and Cdk2). Moreover, ORM repressed the expression of HPV E6/ E7 oncoproteins and restored the expression of their downstream target tumor suppressor proteins (p53, Rb and PTPN 13). As a result, ormeloxifene induces radio-sensitization in cervical cancer cells and caused potent tumor growth inhibition in orthotopic mouse model. Taken together, ormeloxifene represents an alternative therapeutic modality for cervical cancer which may have rapid clinical translation as it is already proven safe for human use.

Figure 1

Ormeloxifene inhibits cell proliferation and motility. (A) Ormeloxifene decreases cellular proliferation of Caski and SiHa cells. Caski and SiHa cells were treated with ormeloxifene (10, 20, 25 µM) for 48 hours and MTS method was used to determine proliferation and absorbance was measured at 490 nm. Results were normalized to the vehicle control (ETOH). Error bars show SEM, n = 3. *p < 0.05. (B) Growth kinetics through xCELLigence RTCA. Caski and SiHa cell lines were treated with 20 µM ormeloxifene and growth kinetics (rate of real time proliferation) was measured. (C,D) Ormeloxifene inhibits clonogenic potential of cells. (C) Cells showed inhibited colony forming ability after 15 days of ormeloxifene treatment. Results were normalized to the ETOH control. Error bars show SEM, n = 3. *p < 0.05. (D) Qualitative representation of inhibited clonogenecity of cells. Images were taken at 200X. (E) Ormeloxifene decreased the cellular migration and invasion. Cells were treated with ormeloxifene for 24 hours and images were taken at 100X. Both cells show clear inhibition of motility and invasion confirmed by Boyden chamber method. (F) Motility kinetics through xCELLigence RTCA. Real time migratory and invasive properties of Caski and SiHa cells were also confirmed using xCELLigence system.

Figure 2

Ormeloxifene induces apoptosis and arrests cell cycle of cervical cancer cells. Ormeloxifene decreases mitochondrial membrane potential (MMP). (A) Cells were stained with TMRE dye for 20 mins at 37 °C and next treated with ormeloxifene for 24 hours to detect the healthy mitochondria. Representation of qualitative images showed decreased TMRE stain signifying reduced MMP, images were taken at 200X. (B) Flow cytometry results also showed a reduction in MMP (decreased TMRE fluorescence level). Results were normalized to the ETOH control. Error bars show SEM, n = 3. *p < 0.05. Ormeloxifene induces apoptosis. (C) Cells were treated with ormeloxifene for 24 hours and analyzed by flow cytometry using Annexin V and 7AAD dyes. (D) Graphical representation of flow cytometry data for Annexin V positive cells (early apoptosis). Error bars show SEM, n = 3. *p < 0.05. (E) Generation of reactive oxygen species (ROS). Cells were treated with 25 µM ormeloxifene and stained with DCFH-DA dye. Flow cytometry data represented an elevated levels of DCFH-DA dye which denotes generation of ROS. Error bars show SEM, n = 3. *p < 0.05. (F) Cell cycle was arrested at G1-S transition. Cells were treated with ormeloxifene for 24 hours, stained with PI dye and analyzed by flow cytometer and ModFit software for cell cycle analysis. ± shows SEM, n = 3. *p < 0.05.

Figure 3

Ormeloxifene modulates the expression of cell cycle regulatory proteins and decreases PI3K/Akt pathway. (A) Immunoblots for protein expression. Caski cells were treated with ormeloxifene for 24 hours and immunoblots were preformed to detect p21, CdK2, Cyclin E, Akt, pAkt and PI3K proteins. β -actin was used as a loading control. All full-length blots are presented in Supplementary Information. Band quantifications of (B) p21, (C) Cdk2, (D) Cyclin E, (E) Akt, (F) pAkt, and (G) PI3K. Band quantitation was done by using GelQuant software. Band intensity was normalized to β -actin and scaled to the ETOH control. Bars = Relative Expression Level, Error bars show SEM, n = 3. *p < 0.05.

Figure 4

Effects of ormeloxifene on HPV infection. Ormeloxifene decreases (A) HPV E6 and (B) E7 mRNAs. Caski cells were treated with ormeloxifene for 6 hours and mRNA levels of HPV E6 and E7 were determined by quantitative PCR. Expression levels were normalized to a house keeping gene (β-2-Microgloblin) and scaled to the vehicle control (ETOH). Bars = Relative Expression Level, Error bars show SEM, n = 3. *p < 0.05. (C) Ormeloxifene inhibits HPV E6 and E7 proteins and upregulates tumor suppressor proteins. Caski cells were treated with ormeloxifene for 24 hours and western blots were preformed to detect HPV E6 and E7, wt-p53, Rb and PTPN13 proteins. β -actin was used as a loading control. All full-length blots are presented in Supplementary Information. Band quantifications of (D) E6, (E) E7, (F) wt-p53, (G) Rb and (H) PTPN13. Band quantitation was done by using GelQuant software. Band intensity was normalized to β -actin and scaled to the ETOH control. Bars = Relative Expression Level, Error bars show SEM, n = 3. *p < 0.05.

Figure 5

Ormeloxifene sensitizes Caski cells to radiation. Ormeloxifene radio-sensitizes cells and decreases cell proliferation. (A) Caski cells were pretreated with 10 µM ormeloxifene for 6 hours and next exposed to 4Gy radiation. Live cells were counted using a coulter counter. Error bars show SEM, n = 3. *p < 0.05. (B) Representative images of ormeloxifene and radiation treated cells. Images were taken at 100X. Ormeloxifene radio-sensitizes cells and decreases colony forming ability. (C) Caski cells were pretreated with 2, 4, 6 and 8 µM ormeloxifene for 6 hours and next exposed to 4Gy radiation. After 14 days, cells with combination treatment showed fewer colonies than compared to ormeloxifene and radiation alone. Error bars show SEM, n = 3. *p < 0.05. (D) Images represent inhibited clonogenic potential. Images were taken at 200X.

Figure 6

Ormeloxifene inhibits tumor growth in cervical cancer orthotopic mice model. Orthotopic mice model was generated by injecting Caski cells direct into the cervix of female nude mice. (A) Images represent mice from different treatment groups. (B) Ormeloxifene increased overall life expectancy and mice survival than compared to its vehicle control PBS. (C,D) Ormeloxifene also showed a marked reduction in tumor volume and weight than compared to PBS. Error bars show SEM, n = 6. *p < 0.05.