Interleukin-2, interferon-alpha and medroxyprogesterone acetate in metastatic renal cell carcinoma

BACKGROUND: Interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) are the main immuno-biological agents used in the therapy of metastatic renal cell carcinoma (RCC). Unfortunately the promising results obtained in biological studies have not yet been confirmed in clinical studies. One reason is linked to the immunosuppression of metastatic patients which is caused by macrophage products. IL-6 in particular is considered a growth factor for RCC. Medroxyprogesterone acetate (MPA) may interfere with IL-6 macrophage production, possibly causing a synergistic effect in association with IL-2 and IFN-alpha. Therefore the purpose of our study was to evaluate the toxicity and the efficacy of the association between IL-2, IFN-alpha and MPA. PATIENTS AND METHODS: Forty-two consecutive patients with metastatic RCC were enrolled. IL-2 was administered subcutaneously at doses of 4.5 million UI on days 1-5, 8-12, 15-19 and 22-26; IFN-alpha was administered s.c. at a dose of 3 million t.t.w; MPA was administered orally at a dose of 1000 mg daily. This schedule was repeated after a rest of 2 weeks. RESULTS: Toxicity was mild: the main symptoms observed were fatigue and fever. Six CR (14%), five PR (12%), thirteen SD (31%) and seventeen PD (41%) were observed for an overall response rate of 26%. Patients with good PS and low levels of CRP had a better prognosis. CONCLUSION: Considering both the good activity and the low toxicity of this scheme, we think that it could be carried out in normal clinical practice.     

Enhancement of Ad5-TRAIL cytotoxicity against renal cell carcinoma with histone deacetylase inhibitors

Renal cell carcinoma (RCC) will cause greater than 12,000 deaths in the United States this year. The lack of effective therapy for disseminated RCC has stimulated the search for novel treatments including immunotherapeutic strategies, but poor therapeutic responses and marked toxicity have limited their use. The tumor necrosis factor (TNF) family member TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L induces apoptosis in various tumor cell types, while having little cytotoxicity against normal cells. In this study, we investigated the tumoricidal potential of a recombinant adenovirus encoding human TNFSF10 (Ad5-TRAIL), alone and in combination with a panel of histone deacetylase inhibitors (HDACi), against the TRAIL/Apo-2L-resistant RCC line 786-O and normal human renal proximal tubule epithelial cells (RPTEC). Ad5-TRAIL was unable to induce apoptosis in either 786-O or RPTEC alone; however, tumor cell apoptosis occurred when Ad5-TRAIL was combined with HDAC inhibition. Except when combined with trichostatin A, RPTEC were not sensitized to Ad5-TRAIL by HDACi. In 786-O, HDAC inhibition induced CAR expression, permitting increased adenoviral infection and transgene expression. It also induced TRAIL-R2 expression, accelerated the death-inducing signaling complex formation and enhanced caspase-8 activation. Our results demonstrate the utility of combining Ad5-TRAIL with HDACi against RCC, and mechanistically define how this combination modulates RCC sensitivity to TRAIL/Apo-2L and adenoviral infection.     

Enhancement of fluorinated pyrimidine-induced cytotoxicity by leucovorin in human colorectal carcinoma cell lines

Reduced folates have been shown to increase the cytotoxicity of 5-fluorouracil (5-FU) by stabilizing the 5-fluoro-2'-deoxyuridine-5'-monophosphate-thymidylate synthase complex, thus increasing the block in the DNA synthetic pathway. Using an in vitro colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cytotoxicity assay, we tested the effects of 5-FU and 5-fluoro-2'-deoxyuridine (FdUrd) with and without leucovorin (LV) on a panel of 11 human colorectal carcinoma cell lines. The effect of LV on 5-FU and FdUrd was quantitatively similar. A clinically achievable level of LV (20 microM) increased the cytotoxicity in all three replicate experiments in 10 of the 11 cell lines (P less than .05, binomial test). LV alone at a concentration of 20 microM had no effect on cell survival. In three cell lines, 50% inhibition of growth occurred at a clinically achievable area under the curve of 5-FU alone. With the addition of LV, one additional cell line showed 50% growth inhibition at a clinically achievable level of 5-FU. Hence large clinical trials may be necessary to detect a significant improvement in survival as a result of adding LV to the fluorinated pyrimidines.     

Enhancement of Fas-mediated apoptosis in renal cell carcinoma cells by adriamycin

Anti-Fas monoclonal antibody (mAb) kills Fas-expressing cells by apoptosis. Several anticancer agents also mediate apoptosis and may share common intracellular pathways leading to apoptosis with Fas. Thus, we reasoned that combination treatment of drug-resistant cells with anti-Fas mAb and drugs might overcome their resistance. We investigated whether anticancer agents enhance Fas-mediated apoptosis and cytotoxicity against renal cell carcinoma (RCC) cells. Treatment of ACHN RCC cells with anti-Fas mAb in combination with 5-fluorouracil, vinblastine, IFN-alpha, or IFN-gamma did not overcome resistance to these agents. However, combination treatment with anti-Fas mAb and Adriamycin (ADR) resulted in a synergistic cytotoxic effect. Furthermore, synergy was also obtained even when the exposure time was shortened from 24 h to 8 or 2 h. Synergy was also achieved in four other RCC cell lines and five freshly derived human RCC cells. Treatment with anti-Fas mAb in combination with epirubicin or pirarubicin also resulted in a synergistic cytotoxic effect on ACHN cells. Similar results were achieved with a combination of humanized anti-Fas mAb and ADR. Incubation of ACHN cells with ADR augmented the expression of Fas and p53, but not Bcl-2, Bax, or caspase-3. However, the activity of caspase-3 itself was apparently enhanced after treatment with ADR alone or combined treatment with anti-Fas mAb. The synergy obtained in cytotoxicity with anti-Fas mAb and ADR was also achieved in apoptosis. Exposure of ACHN cells and freshly derived RCC cells to ADR enhanced their susceptibility to lysis by peripheral blood lymphocytes and tumor-infiltrating lymphocytes. This study demonstrates that combination treatment of RCC cells with anti-Fas mAb and ADR might overcome their resistance. The sensitization required a low concentration of ADR and a short exposure time, thus supporting the potential in vivo application of a combination of ADR and anti-Fas mAb or immunotherapy in the treatment of ADR- and/or immunotherapy-resistant RCC.     

Phase II trial of pegylated interferon-alpha 2b in patients with advanced renal cell carcinoma

BACKGROUND: Pegylated interferon (IFN) has a longer serum half-life compared with standard IFN, and this allows for weekly dosing. In this study, the efficacy and toxicity of pegylated IFN was assessed in patients with metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: Thirty-two patients with previously untreated mRCC were treated with pegylated IFN-alpha 2b in a prospective, single-arm phase II trial. Pegylated IFN was given by subcutaneous administration on a weekly schedule at a dose of 4.5 microg/kg. RESULTS: Of the 32 assessable patients, 29 (91%) had a nephrectomy previously, and none had been treated previously with systemic therapy. Forty-one percent had good-risk, 53% had intermediate-risk, and 6% had poor-risk features per Memorial Sloan-Kettering Cancer Center risk criteria. The best response was a complete response (CR) in 1 patient (3%). Nine patients (28%) had a partial response. Fifteen patients (47%) had stable disease. The median progression-free survival (PFS) was 5 months (95% CI, 3-7 months), and median overall survival was 31 months (95% CI, 18 months to not reached). Five patients had a prolonged PFS of > or = 17 months, 1 of whom achieved a CR. There were no grade 4 toxicities; primary grade 3 toxicities were hematologic (11 of 32 patients; 34%) and fatigue (4 of 32 patients; 13%). CONCLUSION: Pegylated IFN administered weekly has antitumor activity in patients with mRCC with predominantly good- and intermediate-risk features. This study suggests comparable efficacy and safety compared with standard IFN-alpha.     

Subcutaneous administration of interleukin-2 and interferon-alpha 2b in advanced renal cell carcinoma: long-term results

Clinical data have supported the combination of subcutaneous r-interleukin-2 (rIL-2) and r-interferon-alpha (rIFN-alpha) as a promising combination for advanced renal cell carcinoma (RCC), with a reduced toxicity. We evaluated the activity and safety of this outpatient immunotherapy and report on the clinical results and the long-term survival analysis. Objective responses was observed in 9 of 50 (18%) patients, 6 of whom (12%) achieved a complete response. Overall median survival is 12 months, six patients were surviving at a median follow-up of 24 months, and three (6%) are still progression-free.     

Enhancement of radionuclide induced cytotoxicity by 2-deoxy-D-glucose in human tumor cell lines

The efficacy of targeted radiotherapy can be enhanced by selective delivery of radionuclide to the tumors and/or by differentially enhancing the manifestation of radiation damage in tumors. Our earlier studies have shown that the 2-deoxy-D-glucose (2-DG), an inhibitor of glucose transport and glycolytic ATP production, selectively enhances the cytotoxicity of external beam radiation in tumor cells. Therefore, it is suggested that 2-DG may also enhance the cytotoxic effects of radionuclides selectively in tumor cells, thereby improving the efficacy of radionuclide therapy. In vitro studies on breast carcinoma (MDA-MB-468) and glioma (U-87) cell lines, has been carried out to verify this proposition. Clonogenicity (macrocolony assay), cell proliferation, cytogenetic damage (micronuclei formation) and apoptosis were investigated as parameters of radiation response. Mean inactivation dose D (dose required to reduce the survival from 1 to 0.37), was 48 MBq/ml and 96 MBq/ml for 99 mTc, treated MDA-MB-468 and U-87, respectively. The dose response of growth inhibition, induction of micronuclei formation and apoptosis observed under these conditions, were correlated well with the changes in cell survival. Presence of 2-DG (5 mM) during radionuclide exposure (24 hrs), reduced the survival by nearly 2 folds in MDA-MB-468 (from 48.5 MBq to 18.5 MBq) and by 1.6 folds in U-87 cells (from 96 MBq to 66 Mbq). These results clearly show that the presence of 2-DG during radionuclide exposure, significantly enhances the cytotoxicity, by increasing mitotic as well as interphase death. Further studies to understand the mechanisms of radio-sensitization by 2-DG and preclinical studies using tumor-bearing animals, are required for optimizing the treatment schedule.     

Defective Jak-Stat activation in renal cell carcinoma is associated with interferon-alpha resistance

Chemotherapy is ineffective against metastatic renal cell carcinoma (RCC). Interferon (IFN)-alpha has become the most common agent used in clinical therapy to overcome this malignant tumor, although a satisfactory response has not been achieved and the mechanism of resistance of RCC to IFN-alpha remains unclear. The purpose of the present study was to evaluate the susceptibility of RCC cells to IFN-alpha and clarify the mechanism of IFN-alpha resistance in RCC. Six RCC cell lines and three types of IFN-alpha were used, and the expression, activation and effects of transfection of possible proteins or factors reported to be involved in IFN-alpha signaling were examined to clarify the mechanism of resistance. The results suggest that the resistance of RCC to IFN-alpha is associated with the lack of Jak1, Tyk2 and Stat1 expression and defective Jak-Stat activation, but not with a lack of IFN-alpha receptor, suppressors of cytokine signaling induction or other factors examined. Moreover, phosphorylation of Jak-Stat pathway components and reversion of IFN-alpha resistance in RCC were observed upon transfection with Jak1, Tyk2 or Stat1 vector. These results suggest that restoring the expression of Jak or Stat1 might strikingly increase the susceptibility of RCC to IFN-alpha and may be a new strategy for improving the response of RCC to IFN-alpha treatment. The Jak-Stat pathway should therefore be an appropriate target for the treatment of RCC.     

Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by L-buthionine sulfoximine

Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemic patients and apoptosis in various tumor cells in vitro. To develop As2O3-based combination chemotherapy for renal cell carcinoma (RCC), we investigated the cytotoxic effects of As2O3 in combination with chemotherapeutic agents or L-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor. Cytotoxicity and synergy were assessed by the MTT assay and isobolographic analysis, respectively. Apoptosis was monitored by Hoechst 33342 staining, flow cytometrical analysis, and DNA fragmentation assay. Treatment of ACHN cells with As2O3 in combination with adriamycin, vinblastine, or 5-fluorouracil induced an antagonistic effect. However, combination treatment with As2O3 and BSO resulted in a synergistic cytotoxic effect. Synergy was also obtained in Caki-1, Caki-2, NC65 cells and freshly derived RCC cells from 6 patients. Simultaneous treatment of ACHN cells with As2O3 and BSO caused significantly more cytotoxicity than the As2O3 first BSO second or the reverse treatment. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of As2O3 and BSO was realized by inducing apoptosis. This combination markedly decreased intracellular GSH content and GSH-S-transferase (GST) activity. However, neither the intracellular GSH nor GST was decreased by As2O3 with adriamycin, vinblastine, or 5-fluorouracil. Furthermore, the GSH-increasing agents N-acetylcysteine and lipoic acid significantly inhibited the combined cytotoxicity of As2O3 and BSO. These findings indicate that BSO sensitizes RCC cells to As2O3-induced apoptosis through the down-regulation of the intracellular GSH redox system, suggesting the potential application of a combination of As2O3 and BSO for the treatment of RCC.     

Connexin 32 potentiates vinblastine-induced cytotoxicity in renal cell carcinoma cells

We have reported that connexin (Cx) 32 gene, a member of gap junction protein family, acts as a tumor suppressor gene in human renal cell carcinoma (RCC). Of solid tumors, RCC is one of the most chemoresistant cancers, and there is no effective cancer chemotherapy against RCC at present. In this study, we examined if the combination of Cx32-dependent tumor-suppressive effect and vinblastine (VBL), a chemotherapeutic agent which has been utilized for clinical RCC treatment, could be effective in enhancing the sensitivity of RCC to VBL treatment. Cx32 expression in a human metastatic RCC cell (Caki-1 cell) significantly enhanced in vitro and in vivo VBL-induced cytotoxicity on the cell. Cx32 expression in the RCC cells potentiated VBL-induced apoptosis compared to the Cx32-negative RCC cells in vitro as well as in vivo. The enhancing apoptosis in the RCC cells by Cx32 mainly depended on the decrease of P-glycoprotein (P-gp), a multidrug resistance gene-1 (MDR-1) product responsible for reduction of VBL accumulation into the cells. We also observed that silencing of Cx32 by short interfering RNA (siRNA) treatment elevated the level of P-gp in Caki-1 cells and that inhibition of P-gp function enhanced VBL-induced apoptosis in the RCC cells. These results suggest that Cx32 is effective to enhance VBL-induced cytotoxicity in Caki-1 cells via the reduction of P-gp. Overall, it seems that the combination of Cx32-dependent tumor-suppressive effect and VBL is promising as a new cancer therapy against RCC.     

Daily subcutaneous ultra-low-dose interleukin 2 with daily low-dose interferon-alpha in patients with advanced renal cell carcinoma.

A limited institution Phase II pilot study was performed using a very low-dose combination of daily s.c. interleukin (IL)-2 with IFN-alpha-2b in patients with advanced renal cancer in an attempt to duplicate or increase the response documented with higher dose schedules without the attendant toxicity profile. We selected a dose of IL-2 with documented immunological activity and combined it with clinically active low-dose IFN. Between August 1994 and September 1996, 19 patients with metastatic renal cell carcinoma, who had been judged incapable of tolerating high-dose i.v. IL-2, were treated with IL-2 (1 million units/m2/day) and IFN (1 million units/day), administered s.c. daily. All treatments were administered on an outpatient basis. Virtually all patients had bulky tumor burden with multiple sites of involvement, including five patients with bone metastases. No major objective responses were observed; however, one patient experienced a minor response lasting 13 months, with an associated improvement in performance status. Median survival was 6 months, and 1-year survival was 16%. Toxicity was generally mild and consisted almost entirely of constitutional symptoms. No serious grade 3 or 4 toxicity was observed, although two patients withdrew from treatment due to treatment-related fatigue. On therapy, mild eosinophilia but no lymphocytosis was noted; in fact, peripheral lymphocyte counts decreased, only to rebound after treatment was discontinued. No toxic deaths occurred. Despite the reasonable tolerability of this daily low-dose s.c. regimen, we conclude that this regimen is an ineffective treatment in metastatic renal cell carcinoma patients who are incapable of tolerating high-dose i.v. IL-2.     

Enhancement of cisplatin cytotoxicity by SAHA involves endoplasmic reticulum stress-mediated apoptosis in oral squamous cell carcinoma cells

Purpose  The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), enhances cisplatin [cis-diammine dichloroplatinum (II)] (CDDP)-induced apoptosis in the oral squamous cell carcinoma (OSCC) cell line by complex, multifunctional mechanisms. We investigated the role of endoplasmic reticulum (ER) stress in the enhancing effect of SAHA on CDDP, compared with the ER stressor thapsigargin. Methods  We chose OSCC cell line HSC-3 to ascertain the mechanism of SAHA-enhanced cytotoxicity among various cell lines. HSC-3 cells were incubated with CDDP/SAHA for 48 h, followed by the assessment of cell chemosensitivity to CDDP with MTT and TUNEL assays. Western blot analysis was used to detect the expressions of ER-related molecules, and flow cytometry was used to monitor caspase activity. Results  Treatment with CDDP/SAHA potently induced apoptosis in HSC-3 cells with a significant increase in caspase-4 and -12 functions. For example, 60% of cells became apoptotic after 48 h of treatment with CDDP/SAHA. In addition, SAHA alone rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)α, which is up-regulated during ER stress. Inhibition of ER stress by salubrinal, an inhibitor of eIF2α dephosphorylation, abrogated SAHA’s enhancement of CDDP cytotoxicity. Levels of phospho-Akt are decreased in SAHA-treated cells, and this is in turn associated with increased activity of protein phosphatase 1 (PP1) by SAHA, the phosphatase upstream of Akt. Conclusion  These data indicate that up-regulation of specific-ER stress-associated events is an integral part of the mechanism by which SAHA enhances CDDP-induced apoptosis, and PP1 up-regulation followed by Akt dephosphorylation plays an important role in SAHA-enhanced CDDP apoptosis.     

Enhancement of 5-fluorouracil cytotoxicity on human colon cancer cells by retrovirus-mediated interferon-alpha gene transfer.

Gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Several reports documented the clinical effects of interferon-alpha (IFN-alpha) in management of patients with advanced colorectal carcinoma. We report for the first time, the successful transduction of human IFN-alpha gene into colon cancer cells, COLO 201 using a replication-defective retroviral vector. Retrovirus-containing supernatant from PA 317 packaging cells was used to infect colon cancer cells, COLO 201 and NIH 3T3 cells. Transient infection showed that cell proliferation and cell viability were significantly suppressed in colon cancer cells transduced with IFN-alpha gene. Moreover, IFN-alpha-transduced cells acquired less resistance to 5-FU induced apoptosis. These data demonstrate that IFN-alpha gene transfer may have a clinical application and can be combined with chemotherapy for treatment of advanced colorectal cancer.     

Lack of interleukin-2 (IL-2) expression and selective expression of IL-10 mRNA in human renal cell carcinoma

Freshly isolated tumor-infiltrating lymphocytes (TIL) are often functionally deficient. Since one of the key functional parameters of an immune response is the local production of cytokines, we studied the expression of cytokine genes in freshly isolated renal cancer tissue. Using a PCR-assisted mRNA amplification assay, the constitutive expression of mRNA for 10 different cytokines was assessed in renal cancer tissue. We compared the cytokine mRNA expression in freshly isolated samples of renal carcinomas, renal cancer cell lines established from the tumor samples, peripheral blood mononuclear cells (PBMC) and non-tumor kidney tissue isolated from the same patients. IL-10 mRNA expression was detected only in tumor samples, while renal cancer lines, PBMC and non-tumorous kidney tissues were devoid of this cytokine. One-third of the tumor samples but none of the normal kidney samples also expressed G-CSF mRNA. IL-6, TNF-α and IFN-γ mRNA were expressed non-selectively in tumors, PBMC and normal renal tissue. Expression of IL-2, IL-3 and IL-4 mRNA was not detected in any of the tissues analyzed. Established renal cancer lines exhibited expression of IL-lα, IL-6, TNF-α and GM-CSF. Culture of tumor-derived T cells with anti-CD3 monoclonal antibody (MAb) resulted in expression of IL-2, IL-3 and IL-4 mRNA. In contrast, none of these cytokines was detected in culture with recombinant human IL-2 alone. Since IL-10 is known to suppress antigen presentation, these findings have important implications for the possible in vivo role of IL-10 as a suppressor of local anti-tumor response.     

Successful treatment for metastases from renal cell carcinoma with alternation of interferon-alpha subtypes

Here we present a case in which alternation of interferon-alpha (IFN-α) treatments was effective in treating pulmonary metastases and lymph node metastases from renal cell carcinoma (RCC). A 56-year-old man underwent left radical nephrectomy under the diagnosis of left RCC. The histological diagnosis was clear cell carcinoma G2, IFN-α, pT1b. He subsequently underwent two operations for right pulmonary metastasis and right hilar lymph node metastasis. Postoperatively he was treated with intramuscular administration of natural IFN-α (Sumiferon) which prevented definite recurrence for 1 year. However, multiple pulmonary metastases and left hilar lymph node metastasis occurred 11 months after discontinuation of Sumiferon. Therefore, treatment with another natural IFN-α (OIF) was started. Although OIF was continued for 7 months, pulmonary metastases and left hilar lymph node metastasis continued to progress. Therefore, treatment was changed to Sumiferon, after which the pulmonary metastases and left hilar lymph node metastasis decreased in size. The metastases showed no progression for 16 months after switching from OIF to Sumiferon.