Selected Clinical and Pathological Features
The age of patients undergoing hysterectomies for cancer ranged from 45 to 91 years with a mean of 58 years. The mean age of patients with ESC was 65 years (ranged from 58 to 91), while the mean age of patients with EEC was 57 years (ranged from 45 to 77). The average age of patients with positive Nrf2 expression ranged from 50 to 79 years with a mean of 59 years. All but 23 cancer patients presented had postmenopausal bleeding. Among the 23 patients who did not have postmenopausal bleeding, 15 had an abnormal pap smear, 6 had an increased endometrial stripe thickness found by ultrasound, and 2 were found to have a pelvic mass by a routine pelvic examination. Among the benign control group, average patients’ age ranged from 46 to 82 with an average age of 58.
Endometrial Serous Carcinoma had elevated Nrf2 expression
A total of 117 cases including 46 ESC, 51 EEC, and 20 benign endometria were studied. H&E staining was examined to differentiate the type of endometrial tumors. Representative images are shown (, panel a, c, and e). A consecutive tissue section from the same patient was also analyzed for Nrf2 expression by IHC (, panel b, d, and f). Nrf2 expression was scored as positive or negative for each case, according to the criteria described in the materials and methods section. The data are summarized in . Among the 46 ESC cases, 41 (89%) were positive for Nrf2 expression. In contrast, Nrf2 was only expressed in 14 of 51 (28%) EEC cases. The difference in Nrf2 expression was statistically significant between the two types of endometrial cancer (P<0.0001). Furthermore, there was no Nrf2 expression in all 20 benign endometria consisting of 5 atrophic, 5 weakly proliferative, 6 proliferative, and 4 hyperplastic endometria. These results implicate a strong correlation between elevated Nrf2 expression and the aggressiveness of cancer, indicated by higher Nrf2 expression in type-II endometrial cancer tissues. Furthermore, overexpression of Nrf2 correlated with high expression of HO-1, a Nrf2 target gene. HO-1 was highly expressed in ESC tissue sections while there was no expression detected in benign tissue sections (). These data demonstrate that Nrf2 was functional in ESC and was able to activate its downstream genes.
Fig 1 Type II endometrial cancer had elevated Nrf2 expression. (A) The left panels represent H&E staining and the right panels represent IHC staining with a monoclonal Nrf2 antibody. No Nrf2 expression was detected in benign tumors or in endometrial (more ...)
Comparison of Nrf2 protein expression in different types of endometrial lesions.
SPEC-2 cells express a higher level of Nrf2 than Ishikawa cells and are more resistant to chemotherapeutic drugs
To investigate if there is a correlation between basal Nrf2 protein level and resistance to chemotherapeutic drugs two cell lines, Ishikawa and SPEC-2, were used. Ishikawa and SPEC-2 were isolated from tumors of patients with type I (EEC) or type II (ESC) endometrial carcinomas, respectively. Although no difference in Nrf2 mRNA transcription was observed (, Nrf2 panel), the mRNA expression of Nrf2-downstream genes, including NQO1, HO-1, MRP2, GCLC, and GCLM, was increased in SPEC-2 cells (), indicating the activation of the Nrf2-mediated response. Transcription of MRP2 was increased almost 9-fold in SPEC-2 cells compared to that in Ishikawa cells. Interestingly, Keap1 mRNA was decreased about 30% in SPEC-2 cells compared to Ishikawa cells (). Next, the protein level of Nrf2 and two of its downstream genes was measured under both basal and tert-Butylhydroquinone (tBHQ)-induced conditions. SPEC-2 had higher levels of Nrf2, NQO1, and γ-GCS compared to Ishikawa under both basal and induced conditions (, compare lane 1 to 3 and lane 2 to 4). Consistent with a decrease in Keap1 mRNA, there was a decrease in Keap1 protein level in SPEC-2, compared to Ishikawa (, Keap1 panel and the bar graph). Next, the sensitivity of these two cell lines to cisplatin and paclitaxel, two commonly used chemotherapeutic drugs for treating endometrial carcinoma, was measured. Ishikawa cells were more sensitive to cisplatin and paclitaxel treatment than SPEC-2 (). Collectively, these data establish a correlation between the level of Nrf2 and resistance to chemotherapeutic drugs.
Fig 2 SPEC-2 cells express a higher level of Nrf2 than Ishikawa and are more resistant to chemotherapeutic drugs. (A) mRNA levels of Nrf2, Keap1, NQO1, HO-1, MRP2, GCLC, and GCLM were compared between Ishikawa and SPEC-2 cells using real-time RT-PCR. The data (more ...)
Transient silencing of endogenous Nrf2 enhanced the sensitivity of SPEC2 to chemotherapeutic drugs
To further explore the role of Nrf2 in chemoresistance, Nrf2 was knocked down by transient transfection of Nrf2-siRNA into Ishikawa and SPEC2 cell lines. As shown in , the protein level of Nrf2 was reduced significantly in both cell types, as well as the levels of NQO1 and γ-GCS (, compare lane 1 with 2; lane 3 with 4). Consistent with the results shown in , the basal level of Nrf2 in SPEC-2 cells is higher than that in Ishikawa cells (, compare lane 1 with 3). Next, the effect of Nrf2 knockdown on cell resistance to therapeutic drugs was assessed. Knockdown of Nrf2 in Ishikawa cells did not significantly affect cell viability in response to cisplatin or paclitaxel (, left two panels). In contrast, silencing of Nrf2 in SPEC-2 cells significantly sensitized SPEC-2 cells to cisplatin and paclitaxel (, right two panels). It is conceivable that the substantial effect of Nrf2 knockdown in SPEC-2 is due to the higher basal level of Nrf2 in this cell line, compared to Ishikawa.
Fig 3 Transient knockdown of Nrf2 expression by Nrf2-siRNA sensitized SPEC-2 cells to chemotherapeutic drugs. (A) The protein levels of Nrf2, NQO1, and γ-GCS were compared between Ishikawa and SPEC-2 cells transfected with Nrf2-siRNA or control siRNA. (more ...)
Establishment of a stable SPEC-2 cell line overexpressing Keap1
Transient silencing of Nrf2 sensitized SPEC-2 cells to chemotherapeutic drugs; therefore, SPEC-2 was used to establish a stable cell line, in which Keap1 was overexpressed. Expression of Nrf2 and its downstream genes, as well as their resistance to chemotherapeutic drugs were compared between vector-containing (control) and Keap1-CBD-containing (Keap1+) cell lines. Overexpression of Keap1-CBD did not affect the mRNA level of Nrf2 (, Nrf2 panel), which is consistent with the established notion that Keap1-mediated negative control is primarily at the level of Nrf2 protein stability. Stable incorporation of Keap1 cDNA was confirmed by a 5-fold increase in Keap1-mRNA expression in Keap1+ cells, compared to control cells (, Keap1 panel). As a consequence of Nrf2 inhibition by Keap1-CBD overexpression, the mRNA level of NQO1, HO-1, MRP2, GCLC, and GCLM was significantly reduced in Keap1+ cells compared to control cells (). Immunoblot analysis also showed a reduction in the protein level of Nrf2, NQO1, and γ-GCS in Keap1+ cells under both basal and induced conditions (, compare lane 1 with 2; lane 3 with 4). Consistent with a reduction of Nrf2 in Keap1+ cells, NQO1 activity and glutathione levels were also lower in Keap1+ cells than in control cells (). Collectively, these results demonstrate the successful establishment of a stable cell line with reduced Nrf2 expression. Next, the sensitivity of control and Keap1+ cells to cisplatin or paclitaxel was compared. Keap1+ cells were significantly more sensitive to cisplatin and paclitaxel than control cells (), indicating that Nrf2 protects cells against the cytotoxic effects of cisplatin and paclitaxel.
Fig 4 Stable knockdown of Nrf2 by overexpression of Keap1 increased the susceptibility of SPEC-2 cells to chemotherapeutic drugs. (A) Two SPEC-2-derived cell lines, control and Keap1+, stably expressing the control vector or Keap1-CBD were established using (more ...)
Knockdown of Nrf2 increased chemosensitivity of SPEC-2 xenografts to cisplatin
To further confirm the role of Nrf2 in chemoresistance, a human tumor xenograft model in SCID mice was used. As shown in , the average tumor size developed after injection of control cells or Keap1+ cells without cisplatin treatment was not much different at the termination of the experiment. In contrast, the Keap1+ xenografts responded better to cisplatin than the control xenografts as evidence by the greater size reduction of Keap1+-tumors than control-tumors in the treated groups (). Interestingly, the average tumor weight in the Keap1+ group was less than that in the control group, even in the untreated condition (). It is worth mentioning that Keap1+-tumors in the untreated group were more cystic and some of them lost fluid during their removal from the mice, resulting in an underestimation of tumor weights in the untreated Keap1+-group. Furthermore, the difference in tumor size between the Keap1+/saline and Keap1+/cisplatin groups is significantly larger than that between control/saline and control/cisplatin, with the smallest tumor size observed in the Keap1+/cisplatin group (). Collectively, these results indicate that genetic suppression of Nrf2 expression rendered xenografts more susceptible to cisplatin, demonstrating a critical role of Nrf2 in chemoresistance in vivo.
Fig 5 Keap1+-tumors had a substantial reduction in tumor volume following cisplatin treatment. A total of 32 female SCID mice were divided into four groups (8 mice per group): (i) mice were injected with control cells and treated with saline only; (ii) mice (more ...)
Keap1+ xenografts had an increase in apoptosis and a decrease in proliferation
The excised tumors were used to confirm reduced expression of Nrf2 in Keap1+ tumors. Three tumors excised from three different mice in each group were used for immunoblot analysis. Overexpression of Keap1-CBD was confirmed by immunoblot analysis with an anti-CBD antibody (, Keap1-CBD panel). Consequently, the protein level of Nrf2 was significantly lower in Keap1+-tumors, compared to that in control-tumors in both untreated and treated groups (, compare lane 1–3 to lane 7–9; lane 4–6 to lane 10–12, and the bar graph). IHC data further confirmed inhibition of Nrf2 expression in Keap1+ xenografts, as shown by negative staining of Nrf2 in Keap1+-tumors in both untreated and treated groups (). Next, cellular proliferation in tumor tissues was assessed by IHC with an anti-Ki67 antibody. Cisplatin treatment significantly inhibited proliferation in both control- and Keap1+-tumors (). Furthermore, there were less proliferative cells in Keap1+-tumors, compared to control-tumors following cisplatin treatment (). On the other hand, Keap1+-tumors showed a marked increase in the number of apoptotic cells as detected by TUNEL analysis (). Taken together, these results clearly demonstrate that suppression of Nrf2 expression results in a reduction in tumor cell proliferation and enhanced apoptosis of tumor cells in response to cisplatin treatment, which is likely the basis for sensitization of cancer cells to chemotherapeutic drugs when Nrf2 is inhibited.
Fig 6 Keap1+-tumors had a reduction in cell proliferation and an induction of apoptosis in response to cisplatin treatment. (A) Protein levels of Nrf2 and Keap1-CBD in tumor tissues. Each lane contained the tumor tissue lysate extracted from individual mouse. (more ...)