Progression of renal cell carcinoma is inhibited by genistein and radiation in an orthotopic model

Abstract

Background: We have previously reported the potentiation of radiotherapy by the soy isoflavone genistein for prostate cancer using prostate tumor cells in vitro and orthotopic prostate tumor models in vivo. However, when genistein was used as single therapy in animal models, it promoted metastasis to regional para-aortic lymph nodes. To clarify whether these intriguing adverse effects of genistein are intrinsic to the orthotopic prostate tumor model, or these results could also be recapitulated in another model, we used the orthotopic metastatic KCI-18 renal cell carcinoma (RCC) model established in our laboratory.

Methods: The KCI-18 RCC cell line was generated from a patient with papillary renal cell carcinoma. Following orthotopic renal implantation of KCI-18 RCC cells and serial in vivo kidney passages in nude mice, we have established a reliable and predictable metastatic RCC tumor model. Mice bearing established kidney tumors were treated with genistein combined with kidney tumor irradiation. The effect of the therapy was assessed on the primary tumor and metastases to various organs.

Results: In this experimental model, the karyotype and histological characteristics of the human primary tumor are preserved. Tumor cells metastasize from the primary renal tumor to the lungs, liver and mesentery mimicking the progression of RCC in humans. Treatment of established kidney tumors with genistein demonstrated a tendency to stimulate the growth of the primary kidney tumor and increase the incidence of metastasis to the mesentery lining the bowel. In contrast, when given in conjunction with kidney tumor irradiation, genistein significantly inhibited the growth and progression of established kidney tumors. These findings confirm the potentiation of radiotherapy by genistein in the orthotopic RCC model as previously shown in orthotopic models of prostate cancer.

Conclusion: Our studies in both RCC and prostate tumor models demonstrate that the combination of genistein with primary tumor irradiation is a more effective and safer therapeutic approach as the tumor growth and progression are inhibited both in the primary and metastatic sites.

Figure 1

Generation of KCI-18/IK cell lines. KCI-18 cells were injected in the kidney of nude mice. Tumor cells were isolated from kidney tumors, expanded in culture and reinjected into mice kidneys. Three serial in vivo passages in the kidney resulted in new tumorigenic cell lines KCI-18/IK.

Figure 2

Chromosomal analysis of KCI-18/IK cell lines. Karyotype of KCI-18 cells showing near tetraploid chromosome complement and multiple clonal aberrations.

Figure 3

Histology of KCI-18 kidney tumors and lung metastases. Following implantation of KCI-18/IK cells in the kidney of nude mice; tumors were resected at different time points and processed for histology. Kidney tumor and lung sections were stained with H&E. Panel A: Development of high-grade carcinoma (arrowheads) with sinusoidal vascular pattern in the kidney (×10). Panel B: Kidney tumor morphology distinct from normal kidney tissue tubular morphology (×25). Panel C: Kidney tumor consisting of cells with large pleomorphic nuclei, prominent nucleoli and abundant eosinophilic cytoplasm (×100). Panel D: Metastatic nodules in lung (×25). Panels E, F: Kidney tumor sections immunostained for cytokeratin (E) and vimentin (F) showing positive cytoplasmic staining for both markers (×50). Magnifications (×-fold) are shown for each picture.

Figure 4

Treatment of KCI-18 kidney tumors with genistein and radiation. Panel A: Design of X-ray irradiation of tumor-bearing kidney. Mice in 3 jigs were positioned on an aluminum frame mounted on the X-ray machine. Exposure of the right kidney is confirmed by metallic clips surgically placed in the kidney and visualized in double exposure X ray radiograph while rest of their body was shielded with lead shield positioned above the mice. Panel B: Treatment schedule diagram. On day 12–14 after KCI-18 cell injection in kidney, mice were treated daily with oral genistein at 5 mg/day/mouse. On day 15, established renal tumors were irradiated with 8 Gy photons. One day later, genistein treatment was resumed and given every other day. On day 28, mice were killed and tumors and metastatic tissues were resected. Panel C: Response of primary kidney tumors to radiation and genistein. Weights of the tumor-bearing kidneys and their median weight, from 7–9 mice per treatment group, are reported in boxplot showing the range of data. Panel D: Response of mesentery metastases. Mesentery nodules were prominent along the intestines and enumerated. Each dot represents the number of metastatic nodules per mouse. Panel E: Response of lung metastases. White metastatic nodules were enumerated on lungs insufflated with India ink and bleached as described in Materials and Methods. Each dot represents the number of nodules per mouse. Data from four experimental treatment groups are shown: mice treated with vehicle (Control), genistein only (Gen), radiation only (Rad), genistein combined with radiation (Gen+Rad).

Figure 5

Histology of KCI-18 kidney tumors treated with genistein and radiation. Kidney tumors, resected from mice of the experiment described in Figure 4, were processed for histology and tumor sections were stained with H&E. The main findings were labeled on the prints with T for tumor, V for vessel, M for mitosis, H for hemorrhages, DG for degenerative, N for necrosis, A for apoptosis, F for fibrosis, DC for detached cells, R for rhabdoid cells, NKT for normal kidney tissue, IF for inflammatory cells and AM for abnormal mitosis. Panels A, B: Kidney tumor from control mice showing high-grade and very vascularized carcinoma (A, ×50) with frequent mitosis (B, ×100). Panels C, D: Kidney tumor from mice treated with genistein showing extensive hemorrhages (C, ×50), degenerative changes in tumor cells, apoptotic cells and areas of necrosis (D, ×100). Panels E, F: Irradiated kidney tumor, with areas of tumor destruction, showing fibrosis and apoptotic cells (E, ×50), focal areas with atypical detached rhabdoid cells (F, ×100). Panels G, H: Kidney tumor from mice treated with genistein and radiation, showing smaller residual tumor area adjacent to normal kidney tissue (G, ×25). The residual tumor looked hemorrhagic and consisted of large areas of detached rhabdoid cells, atypical giant cells with large nuclei and inflammatory cells (H, ×100). Lower and higher magnifications (×-fold) are presented to both show wider areas of tumor histology and focus on major findings.

Figure 6

Histology of mesentery tumor nodules following treatment with genistein. Sections of the mouse bowel were stained with H&E. Panel A: Section of bowel (B) from mice treated with genistein showing tumor nodules (TN) in mesentery adipose tissue (AD). Panel B: Section of bowel (B) from mice treated with genistein and radiation and adipose tissue (AD) showing normal morphology of mesentery lining the bowel. Magnifications, ×25.