Verteporfin exhibits YAP-independent anti-proliferative and cytotoxic effects in endometrial cancer cells - PubMed (original) (raw)

Verteporfin exhibits YAP-independent anti-proliferative and cytotoxic effects in endometrial cancer cells

Venkata Ramesh Dasari et al. Oncotarget. 2017.

Abstract

Endometrial Carcinoma (EMCA) is the most common gynecologic malignancy and the fourth most common malignancy in women in the United States. Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. Verteporfin (VP), a benzoporphyrin derivative, was identified as an inhibitor of YAP-TEAD interaction. We investigated the therapeutic efficacy and mechanism of VP in EMCA. The efficacy of VP on cell viability, cytotoxicity and invasion was assayed in EMCA cell lines. An organoid model system was also developed to test the effect of VP on apoptotic markers in an in vitro model system. Treatment with VP resulted in a decrease in cell viability, invasion and an increase in cytotoxicity of EMCA cells. These effects occurred as early as 15 minutes following treatment. Similarly, VP treatment versus vehicle control increased apoptosis in human organoid model systems. Quantitative RT-PCR, cDNA based RTPCR array analysis and western blotting were performed to investigate the mechanism of VP action. The cytotoxic and anti-proliferative effects appeared to be independent of its effect on YAP. Our results suggest that VP is a promising chemotherapeutic agent for the treatment of endometrial cancer.

Keywords: YAP; endometrial carcinoma; hippo pathway; organoids; verteporfin.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

Figures

Figure 1

Figure 1. Anti-proliferative effects of VP on EMCA cells

A. Cell Viability assay after treatment of HEC-1-A and HEC-1-B cells (10,000 each well) with DMSO or VP at 10 nM concentrations from 0-3h. Error bars indicate Mean ±SEM. n=4. *Statistically significant at p<0.05. (p = 0.047, 0.0089, and 0.0001 for HEC-1-A and 0.0001, 0.0001 and 0.0001 for HEC-1-B respectively). Experiment is repeated 4 times with at least 3 replicates. B. Trypan blue assay measuring viable cell number at specific time periods. EMCA cells (10,000 each well) were treated with DMSO or VP at 10 nM concentrations after 0-h. Cells that excluded trypan blue were measured. Error bars indicate Mean ±SEM. n=4. *Statistically significant at p<0.05. (p=0.0019, 0.0001, 0.0001, 0.0001 and 0.0001 for HEC-1-A and 0.0017, 0.0001, 0.0001, 0.0001 and 0.0001 for HEC-1-B respectively). Experiment is repeated 3 times with at least 3 replicates. C. LDH cytotoxicity assay of HEC-1-A and HEC-1-B cells after treatment with VP at 10 nM for 0-3h. *Statistically significant at p<0.05. (p=0.0073, 0.00001, 0.0001 for HEC-1-A and 0.0008, 0.0004, 0.0001, 0.0002, 0.00004 for HEC-1-B). Error bars indicate Mean ±SD. n=8. Experiment is repeated 3 times with at least 3 replicates. D. Flow cytometric analysis of EMCA cells after VP treatment for 3h at 10 nM. n=3. Error bars indicate Mean ±SEM. *statistically significant at p<0.05. (p=0.0233, 0.0139 for HEC-1-A and 0.012 and 0.0001 for HEC-1-B respectively). Experiment is done with at least 3 replicates for each cell line.

Figure 2

Figure 2. Inhibition of invasion of EMCA cells by VP

A. EMCA cell lines (each 100,000) were treated with DMSO, VP or untreated at 10 nM for 3h and were allowed to invade through the Matrigel for 36h. Transwell cell inserts with 8μm pores were used. n=9. Bar = 20x. B. Quantitative estimation of matrigel invasion assay. Error bars indicate Mean ±SEM. *Statistically significant at p<0.05. (p=0.0001 for HEC-1-A and 0.0001 for HEC-1-B). Experiment is repeated 3 times with at least 3 replicates for each cell line.

Figure 3

Figure 3. VP induces caspase-3 mediated apoptosis in HEC-1-B Cells and organoids

Confocal images of A. HEC-1-B cells and B. organoid model system (#1077), which were subjected to immunofluorescence detection for cleaved caspase-3 after VP treatment at 10 nM for 3h. Cleaved-caspase-3 (anti-rabbit) is conjugated with goat anti-rabbit Alexa flour secondary antibody. Bar for HEC-1-B = 63x and Bar for organoids is =20x. C. Equal amounts of proteins (40μg) from untreated and treated EMCA cells were loaded on 14% gels and transferred onto nitrocellulose membranes, which were then probed with respective antibodies. GAPDH was used a positive loading control. n=3.

Figure 4

Figure 4. VP downregulates YAP and phospho-YAP of HEC-1-B Cells and organoids

Confocal images of HEC-1-B cells and organoids which were subjected to immunofluorescence detection of A. YAP and B. phospho-YAP after VP treatment. YAP and phospho-YAP are conjugated with goat anti-mouse and goat anti-rabbit Alexa flour secondary antibodies respectively. (A) Upper panel bar=63x and lower panel bar = 20x. (B) Upper panel bar=63x and lower panel bar = 20x. C. Equal amounts of proteins (40μg) from untreated and treated EMCA cells were loaded on 10% gels and transferred onto nitrocellulose membranes, which were then probed with YAP and phospho-YAP (y357) antibodies. GAPDH was used a positive loading control. n=3.

Figure 5

Figure 5. Mechanism of action of VP is independent of YAP in EMCA cells

A. Cell viability assay after treatment of siYAP-HEC-1-B cells (10,000 each well) with VP at 10 nM concentrations after 0h to 3h treatment. Experiment is repeated 3 times with at least 3 replicates for each cell line. Error bars indicate Mean ±SEM. *statistically significant at p<0.05. (p =0.0308, 0.0107 and 0.0051 for respective time points). B. LDH cytotoxicity assay after treatment of siYAP-HEC-1-B cells (10,000 each well) with VP at 10 nM concentrations after 0h to 3h treatment. For all these tests n=3. *Statistically significant at p<0.05. Error bars indicate Mean ±SEM. (p= 0.0493, 0.0317, <0.001 and 0.00002 for siCont-VP and 0.001, <0.0001, <0.0001 for siYAP-VP respectively). Experiment is repeated 3 times with at least 3 replicates for each cell line.

Figure 6

Figure 6. Effect of VP on HIPPO pathway genes of EMCA cells

A. Effect of VP on HIPPO pathway of EMCA cells: cDNA RTPCR arrays were run after treatment of endometrial cancer cells with VP at 10 nM concentration for 3h. Control cells were treated with DMSO (vehicle). n=2 for each cell line and treatment. *Statistically significant at p<0.05. Error bars indicate Mean ±SEM. **B**. RTPCR analysis of selected HIPPO pathway genes. All the genes were normalized to the expression of GAPDH, β-actin, PGK1, LDHA and PPIH. *Statistically significant at p<0.05, by one-way ANOVA. DMSO control vs VP treated samples. Error bars indicate Mean ±SEM. p values are 0.0459, 0.0460 respectively. For each gene, duplicates were performed from 3 different samples for each treatment. n>6. C. Western blot time course of VP effect on YAP-mediated signaling molecules. Equal amounts of proteins (40μg) from untreated and treated (10 nM VP, 0 to 3 hrs.) EMCA cell lysates were loaded on 8% to 10% gels and transferred onto nitrocellulose membranes, which were then probed with respective antibodies. The westerns were run on separate blots. They were reprobed with GAPDH which was used a positive loading control. n=3. D. Western blots showing VP and Lapatinib effects on EGFR-mediated signaling molecules. Equal amounts of proteins (40μg) from untreated and treated (10 nM VP, 15 minutes; Lapatinib 20μM, 1h.) HEC-1-B cell lysates were loaded on 8% to 10% separate gels and transferred onto nitrocellulose membranes, which were then probed with respective antibodies. They were reprobed with GAPDH which was used a positive loading control. n=3.

Similar articles

Cited by

References

    1. American Cancer Society Cancer Facts and Figures. 2016.
    1. Clement PB, Young RH. Non-endometrioid carcinomas of the uterine corpus: a review of their pathology with emphasis on recent advances and problematic aspects. Adv Anat Pathol. 2004;11:117–42. - PubMed
    1. Clement PB, Young RH. Endometrioid carcinoma of the uterine corpus: a review of its pathology with emphasis on recent advances and problematic aspects. Adv Anat Pathol. 2002;9:145–84. - PubMed
    1. Hecht JL, Mutter GL. Molecular and pathologic aspects of endometrial carcinogenesis. J Clin Oncol. 2006;24:4783–91. - PubMed
    1. Weigelt B, Banerjee S. Molecular targets and targeted therapeutics in endometrial cancer. Curr Opin Oncol. 2012;24:554–63. - PubMed

MeSH terms

Substances

LinkOut - more resources