The Mechanisms Involved In The Involution Of Progestin-resistance In Endometrial Carcinoma

Endometrial carcinoma is often diagonised at the average age of sixty-thre years old. 5%-14% of endometrial adenocarcinoma occurs in women (<40 years of age), who very often still desire pregnancy. Progestin, especially medroxyprogesterone acetate (MPA) have been used as conservative treatment in atypical hyperplasia and endometrial adenocarcinoma for a long time.The response rate varies from 57%-75%. It has been reported that conservative treatment followed by IVF enable such patients to obtain a successful pregnancy. These relatively good response rates are encouraging with respect to the possibility of conservative treatment by progestins in young patients who desire for pregnancy, but over a third of patients with endometrial adenocarcinoma will display resistance to endocrine therapies at the time of presentation and most cancer patients that initially respond to progestin treatment would develop resistance later, resulting in tumor progression. Moreover, the conservative treatment failure might delay radical treatment and alter the prognosis. Progestin therapy is limited by the development of resistance in the tumor. The cellular mechanisms of primary and acquired resistance to progestin are poorly understood. In order to investigate the molecular mechanisms we have developed a subcell line resistant to the growth inhibitory effects of progestins from Ishikawa (human endometrial adenocarcinoma cell line) by stepwise selection in increasing concentrations of the synthetic progestin: MPA and investigate the relationship between progestin resistance and the imbalance of ER and PR subtype and the relationship between progestin resistance and the intrinsic/ extrinsic upregulation of the signaling of TGFa/EGF-EGFR.These experiments were divided into 3 parts, as following: Establishment of endometrial carcinoma cell resistant to medroxyprogesterone and its biological characteristic; The imbalance of ER and PR subtype and the intrinsic upregulation of the signaling of TGFa-EGFR might contribute to progestin resistance; D The extrinsic of TGFa/EGF might contribute to progestin resistance.Section Establishment of endometrium carcinoma cell resistant to medroxyprogesterone and its biological characteristicObjective To develop a subcell line of Ishikawa cells resistant to the growth-inhibitory actions of medroxyprogesterone acetate (MPA) and investigate its biological characteristic.Methods Progestin-resistant cell line was developed from Ishikawa human endometrial adenocarcinoma cells by stepwise selection in increasing concentrations of the synthetic progestin, MPA. MTT, FACS and Brdu were used to detect the effect of MPA on the proliferation of the original Ishikawa cell line and the progestin-resistant subcell line. Matrigel invasion assay was used to detect the effect of MPA on the invasive capability of the two cell lines. RT-PCR and Western-blot were used to detect the expression of CyclinD1 under the treatment of MPA over different time. RT-PCR was used to detect the expression of MMP1, MMP9 under the treatment of MPA over different time. Zymographic analysis was used to detect the activity of MMP2, MMP9 secreted from the two cell lines over MPA treatment. Results (1) The progestin-resistant cell line was established from human Ishikawa cell (endometrial cancer cell line) by stepwise selection in continual and increasing concentrations of MPA (1-10μM) over ten months. The doubling time of the progestin-resistant cells (34.18±3.15 hours) grown routinely in the medium containing 10μM MPA was not significantly different from the doubling time of the original Ishikawa cell line (35.14±2.68 hours) grown in the absence of MPA (t=-0.331, P=0.762). (2) MPA inhibited the original Ishikawa cell growth, the inhibitory action was concentration dependent (F=29.525, P=0.000), low concentration of MPA (0.01, 0.1μM) had no effect on the growth of the progestin-resistant cells, but relatively higher concentration (1,10μM) of MPA significantly increased the proliferation of progestin-resistant cells(F=36.20, P=0.00). The inhibitory effects of MPA on the original Ishikawa cells and the stimulatory effects on the progesterone-resistant cells were further confirmed by the cell cycle analysis. The results of cell cycle analysis showed that MPA caused time-dependent inhibition of cell cycle of the original Ishikawa cells, MPA produced a 14.2% fall in the proliferation index on day 1 and a 45% reduction by day 4; reversely, MPA exert no effect on the cell cycle of the progestin-resistant cells on dayl and day 2, by day 3 and day 4, MPA produced an increase in the proliferation index 21.9% and 20.85% respectively. These results were further confirmed by Brdu experiment. (3) Theinvasiveness capability of the progestin-resistant cells were higher than that of the original Ishikawa cells, but the difference was not stastically significant (P>0.05). MPA inhibit the invasiveness capability of the original Ishikawa cells dramatically (t=6.107, P - 0.026) and promote the invasiveness capability of the progestin-resistant cells (t=8.660, P=0.013). (4) Consistent with the phenomena, treatment with MPA on the original Ishikawa cells reduced the expression of CyclinD1, MMP2, MMP9 and the activity of MMP2, and reversely, treatment with MPA on the progestin-resistant cell increased the expression of CyclinD1, MMP2, MMP9 and the activity of MMP2.Conclusions These results suggested that prolonged treatment with MPA on Ishikawa cell could give rise to the resistant effect to MPA, when the resistance was acquired, treatment with MPA could enhance the cancer cell proliferation, invasiveness and metastasis, which might be due to up-regulating CyclinD1, MMP2 and MMP9 expression and the activity of MMP2.Section D The imbalance of ER and PR subtype and the intrinsic upregulation of the signaling of EGFR contribute to progestin resistanceObjective To investigate the relationship between progestin resistance and the imbalance of ER and PR subtype and the relationship between progestin resistance and the intrinsic upregulation of the signaling of TGFa-EGFR. Methods Immunocytochemistry, RT-PCR and western-blot were used to detect the expression of ERa, ERp, PR, PRB and EGFR in the two cell lines. RT-PCR was used to detect the expression of TGFa mRNA in the two cell lines and TGFa expression under the treatment of MPA over different time. Western-blot was used to detect the expression of EGFR-TK in the two cell lines. MTT and FACS were used to detect the effects of EGFR-TK inhibitor AG1478 on the proliferation of the two cell lines.Matrigel invasion assay was used to detect the effects of MPA on the invasive capability of the two cell lines. RT-PCR and western-blot were used to detect the expression of CyclinD1 over AG1478 treatment and RT-PCR was used to detect the expression of MMP2, MMP9 over AG1478 treatment in the two cell lines. Results (1) Compared with original Ishikawa cells, ERa, PRB expression were downregulated and the expression of ERP was upregulated in progestin-resistant cells, the differences were statistically significant (P<0.05). (2) The expression of TGFa and EGFR were higher in progestin-resistant cells than that in the originalIshikawa cells (P<0.05), and the expression of EGFR-TK was higher in progestin-resistant cells than that in the original Ishikawa cells. (3) MPA downregulated the expression of TGFa in original Ishikawa cells, but upregulated the expression of TGFa in progestin-resistant cells, the differences were both statistically significant (P<0.05). (4) MTT results showed that AG1478 inhibited progestin-resistant cells proliferation, the inhibitory effect was dose-dependent (F=18.148,P=0.00; compared with vehicle control, P<0.05), but the inhibitory effects on Ishikawa cells were significant only when AG1478 amounted to a higher level(10μM).The results of cell cycle analysis showed that AG1478 caused inhibition of cell cycle of progestin-resistant cells, the inhibitory rates were 19.4%, 11.2%, 10.7% respectively over 24,48, 72h treatment; but AG1478 produced little fall in the proliferation index on day 1 and 9% reduction on day 2 in the progestin-resistant cells. (5)AG1478 inhibited the invasiveness capability of original Ishikawa cells and the progestin-resistant cells, the inhibitory effects were 42.4% and 58.1% respectively, the inhibitory effect on progestin-resistance cells was higher than the inhibitory effect on original Ishikawa cells(P<0.05). (6) AG1478 downregulated the expression of CyclinD1, MMP2, MMP9 in the two cell lines, the inhibitory effects (55%,97.8%,85.2%) on progestin-resistance cells were higher than the inhibitory effects on Ishikawa cells(23.3%,66.4%,65.2%). Conclusion The downregulation of ERa and PRB, the upregulation of ERβ and the highly activated TGF-EGFR signaling might contribute to progestin resistance in endometrial carcinoma. The inhibitory chemicals AG1478 of EGFR-TK might benefit the conservative treatment of progestin-resistant endometrial carcinoma.Section The extrinsic TGFa and EGF might contribute to progestinresistance in endometrial carcinomaObjective To investigate the relationship between extrinsic growth factors and progestin resistance in endometrial carcinoma.Methods MTT was used to detect the effect of extrinsic growth factors (TGFa/EGF), MPA and TGFa/EGF combined with MPA on the proliferation of Ishikawa cells. Wound healing assays was used to detect the effects of TGFa/EGF, MPA and TGFa/EGF combined with MPA on the metastatic capability of Ishikawa cells. Western-blot was applied to detect the expression of CyclinD1, E-cadherin in Ishikawa cells under the treatment of TGFa/EGF, MPA and TGFa/EGF combined with MPA over different time. RT-PCR and Western - blot were used to investigate the expression of PRB under the treatment of TGFa/EGF and MPA over different concentration and different time. Western-blot was used to detect the expression of P-ERK under different condition that the cells were treated with or without MPA for 48 h and with or without growth factors for the indicated time. Results (1) TGFa/EGF and TGFa/EGF combined with MPA stimulated the proliferation and the motility of Ishikawa cells, contrary to TGFa/EGF, MPA inhibited the proliferation and the motility of Ishikawa cells, compared with control, the differences were statistically significant (P<0.05), the stimulatory effects of TGFa/EGF and TGFa/EGF combined with MPA were not significant(P>0.05). (2) TGFa/EGF and TGFa/EGF combined with MPA increased the expression of CyclinDl and decreased the expression of E-cadherin, MPA decreased the expression of CyclinDl and increased the expression of E-cadherin, these actions were significant when treatment for 24 h. (3) TGFa/EGF and MPA downregulated the expression of PRB, the effects were dose-dependent and these actions were significant when MPA treatment for 12h and TGFa/EGF treatment for 24 h, then PRB expression refreshed but could not attain to the primary level. (4) TGFa/EGF activated MAPK, the expression of P-ERK were higher under the condition that the cells were pretreated with MPA for 48h than the cells not pretreated with MPA with TGFa/EGF for the indicated time.Conclusion (1) TGFa/EGF stimulated Ishikawa cell proliferation, motility and antagonized the inhibitory effect of MPA which might be through upregulating the expression of CyclinDl and downregulating the expression of E-cadherin associated with proliferation and metastasis. (2) MPA inhibited Ishikawa cell proliferation,motility which might be through downregulating the expression of CyclinD1 and upregulating the expression of E-cadherin, but could not antagonized the stimulatory effect of TGFa/EGF on the proliferation and metastasis and the effect of TGFa/EGF on the expression of CyclinDl and E-cadherin. (3) TGFa/EGF downregulated the expression of PRB and attenuated the effect of MPA, and MPA potentiated the stimulatory action of TGFa/EGF on the tyrosine phosphorylation of MAPK. In a word, extrinsic TGFa/EGF accelerated progestin resistance in endometrial carcinoma,.In conclusion, the results of the experiment suggested that prolonged treatment with MPA on Ishikawa cell could give rise to the resistant effect to MPA, the downregulation of ERa, PRB, the upregulation of ERβ and the highly activated intrinsic/ extrinsic TGF-EGFR signaling might contribute to progestin resistance in endometrial carcinoma. When the resistance subtype was acquired, treatment with MPA could enhance the cancer cell proliferation, invasiveness and metastasis, the progestin-resistance cell line progressed from steroid-dependent to growth factors dependent. The inhibitory chemicals of EGFR-TK might benefit the conservative treatment of progestin-resistant endometrial carcinoma.