Interleukin-6 inhibits the growth of prostate cancer xenografts in mice by the process of neuroendocrine differentiation

In vitro, the human prostate cancer (PCA) cell line LNCaP can be permanently transdifferentiated into a quiescent neuroendocrine (NE) phenotype by the cytokine interleukin-6 (IL-6). Recently, we have shown that the growth of prostate cancer cells is significantly suppressed when cocultured with NE cells. In order to explore the inhibitory activity of IL-6 on prostate tumor growth, nude mice bearing xenografts of the PCA cell lines LNCaP and DU-145 (a line that is incapable of NE transdifferentiation by IL-6 in vitro) were treated with IL-6 for 3 weeks, either injected around the tumor or systematically released from implanted minipumps. Both administration forms of IL-6 inhibited the growth of LNCaP xenografts by more than 75% compared to the control group. In contrast, there was no difference in DU-145 tumor growth between IL-6-treated animals and controls. In comparison to control and DU-145 tumors, both IL-6 injected and pump-infused LNCaP tumors exhibited a significant increase in the expression of the NE markers neuron-specific enolase (NSE) and betaIII tubulin. Serum NSE levels were also significantly elevated in both IL-6-treated LNCaP tumor groups when compared to controls. IL-6 treatment resulted in G(0) cell cycle accumulation as evidenced by a loss of Ki-67 expression in > 90% of LNCaP tumor cells. These combined results demonstrate that IL-6-induced NE transdifferentiation of PCA cells has a significant inhibitory effect on tumor growth in mice. Agents, like IL-6, capable of NE transdifferentiation of PCA cells, should be considered as a new therapeutic approach for the treatment of prostate cancer.     

Transfer of a dominant-acting tumor-inducing oncogene from human prostatic carcinoma cells to cloned rat embryo fibroblast cells by DNA-transfection.

The mechanism by which normal human prostate cells develop into prostatic carcinoma cells is not presently known. In the present study we have tested the hypothesis that specific prostatic carcinomas develop as a consequence of activation of a cellular gene(s) with transforming and tumorigenic potential. To test this possibility, high molecular weight DNA was extracted from the human prostatic carcinoma cell line, LNCaP, and cotransfected with a dominant acting neomycin resistance gene, pSV2-neo, into a subclone of Fischer rat embryo fibroblast (CREF) cells, CREF-Trans 6, and NIH-3T3 cells. Cells were selected for growth in G418 and pooled resistant colonies, which were morphologically normal, were injected subcutaneously into athymic nude mice. Tumors developed in several of the animals inoculated with LNCaP DNA-transfected CREF-Trans 6 cells and they were established in monolayer culture. In contrast, no tumors developed in nude mice injected with untransfected CREF-Trans 6 cells, pSV2-neo transfected CREF-Trans 6 cells or LNCaP plus pSV2-neo DNA-transfected NIH-3T3 cells. DNA from the first cycle tumor-derived CREF-Trans 6 cell lines, which were morphologically transformed in monolayer culture, was cotransfected with pSV2-neo a second time into CREF-Trans 6 cells and transfected cells, which were still morphologically normal, were injected into nude mice. Tumors developed in animals and they were again established in tissue culture. Secondary transfectants isolated from animals were morphologically transformed and grew with high efficiency in agar. Both primary and secondary LNCaP-transfected-nude mouse tumor derived-CREF-Trans 6 cells contained human repetitive (Alu) sequences. Although the pattern of Alu integration in the tumor derived CREF-Trans 6 cells were different for different tumors, both primary and secondary tumors contained a single apparently common-sized Alu fragment. The present study indicates that the human prostatic carcinoma cell line, LNCaP, contains a dominant-acting tumor-inducing oncogene which does not induce morphological transformation of CREF-Trans 6 or NIH-3T3 cells in monolayer culture. In addition, the CREF-Trans 6 cell line can detect this tumor-inducing gene function, whereas this activity is not observed in DNA-transfected NIH-3T3 cells.     

Anticachectic effects of the natural herb Coptidis rhizoma and berberine on mice bearing colon 26/clone 20 adenocarcinoma

We previously showed that the natural herb Coptidis rhizoma has an anticachectic effect in nude mice bearing human esophageal cancer cells. We further investigated this phenomenon by examining the anticachectic effect of C. rhizoma in syngeneic mice bearing colon 26/clone 20 carcinoma cells, which cause IL-6-related cachexia after cell injection. We evaluated nutritional parameters such as serum glucose level and wasting of adipose tissue and muscle in tumor-bearing and non-tumor-bearing mice treated with C. rhizoma (CR) supplement or a normal diet. IL-6 levels in those mice were quantified by ELISA and real-time RT-PCR. CR supplementation significantly attenuated weight loss in tumor-bearing mice without changing food intake or tumor growth. Furthermore, these mice maintained good nutritional status. IL-6 mRNA levels in tumors and spleens and IL-6 protein levels in tumors and sera were significantly lower in tumor-bearing mice treated with CR supplement than in those treated with a normal diet. CR supplementation did not affect food intake, body weight, nutritional parameters and IL-6 levels in non-tumor-bearing mice. An in vitro study showed that C. rhizoma and its major component, berberine, inhibited IL-1-induced IL-6 mRNA expression in a dose-dependent manner in colon 26/clone 20 cells. Our results showed that C. rhizoma exerts an anticachectic effect on colon 26/clone 20-transplanted mice and that its effect is associated with tumor IL-6 production. We also suggest that its effect might be due to berberine.     

Enhanced chemosensitization in multidrug-resistant human breast cancer cells by inhibition of IL-6 and IL-8 production

Drug resistance remains a major hurdle to successful cancer treatment. Many mechanisms such as overexpression of multidrug-resistance related proteins, increased drug metabolism, decreased apoptosis, and impairment of signal transduction pathway can contribute multidrug resistance (MDR). Recent studies strongly suggest a close link between cytokines and drug resistance. To identify new targets involved in drug resistance, we established a multidrug-resistant human breast cancer cell line MCF-7/R and examined the cytokine profile using cytokine antibody array technology. Among 120 cytokines/chemokines screened, IL-6, IL-8, and 13 other proteins were found to be markedly increased in drug-resistant MCF-7/R cell line as compared to sensitive MCF-7/S cell line, while 7 proteins were specifically reduced in drug-resistant MCF-7/R cells. Neutralizing antibodies against IL-6 and IL-8 partially reversed the drug resistance of MCF-7/R to paclitaxel and doxorubicin, while a neutralizing antibody against MCP-1 had no significant effect. Inhibition of endogenous IL-6 or IL-8 by siRNA technology significantly enhanced drug sensitivity of MCF-7/R cells. Furthermore, overexpression of IL-6 or IL-8 expression by transfection increased the ADM resistance in MCF-7/S cells. Our data suggest that increased expression levels of IL-6 and IL-8 may contribute to MDR in human breast cancer cells.     

Interleukin 15 augments antitumor activity of cytokine gene-modified melanoma cell vaccines in a murine model

Many studies have demonstrated that interleukin 15 (IL-15) is a cytokine with strong antitumor properties and have suggested its potential use in tumor immunotherapy. IL-15 exerts its effect on innate and acquired immunity with the most prominent action in NK cells and CD8(+) memory T cells. Therefore, many authors have proposed that IL-15 could be a good candidate for augmenting the efficacy of vaccination strategies. In our experiments, in a model of B78-H1 murine transplantable melanoma, tumor-bearing mice were treated with different cytokine-gene modified tumor cell vaccines (producing TNF-alpha, GM-CSF, IL-12 or IL-6/sIL-6R) followed by a series of IL-15 injections. In order to investigate the infiltration of treated tumors by leukocytes, immunohistochemical staining was performed. In every case, the combined therapy was superior to the treatment with either a vaccine or IL-15 alone. Tumors treated with the combination of B78-H1 melanoma cells secreting IL-12 (B78/IL-12 vaccine) and IL-15 were heavily infiltrated by granulocytes. IL-15, either alone or in combination with the B78/IL-12 vaccine, influenced infiltration of tumors with CD3(+) lymphocytes, CD4(+)and CD8(+). To our knowledge, this is the first report that shows the universal genetically-modified tumor cell vaccine-augmenting properties of IL-15. The cytokine can be useful as an adjuvant in cancer gene therapy in humans.     

Interleukin-1 produced by tumorigenic fibroblasts influences tumor rejection.

Oncogene-transformed BALB/c-3T3 fibroblasts which spontaneously or upon immune-activation with cytokines and lipopolysaccharide (LPS) generate IL-1 alpha, were tested for their tumorigenicity as well as their interaction with natural immune defense by NK cells and macrophages. Oncogene-transformed fibroblasts were weakly tumorigenic, since not all mice developed tumors despite application of high doses of tumor cells. This was independent of the immune status of the host. However, in the immunocompetent host those transformed fibroblast lines which spontaneously produced IL-1 alpha grew only transiently and then regressed. After induction of IL-1 alpha production, a decrease in the rate of tumor take was noted and the rate of regression of developing tumors was increased. Regression of IL-1-producing transformed fibroblasts was strongly reduced but not completely abolished in sublethally irradiated mice. This indicated that IL-1 production may predominantly influence T-cell-mediated defense, but some influence on non-adaptive immunity could not be excluded a priori. IL-1 production did not influence susceptibility of transformed fibroblasts towards NK cells and macrophages. However, IL-1-producing transformed fibroblasts were most potent stimulators of NK cells and macrophages, the stimulatory effect being locally restricted. In conclusion, IL-1 producing, oncogene-transformed fibroblasts which generated the cytokine constitutively or upon immune-activation, were rejected from the tumor-bearing host following initial growth. Fibroblast-induced local activation of NK cells and macrophages was shown to play some role in tumor graft rejection. The influence of IL-1 production of transformed fibroblasts on T-cell-mediated defense is addressed in the accompanying report.     

Differences in the expression and effects of interleukin-1 and -2 on androgen-sensitive and -insensitive human prostate cancer cell lines

The presence of interleukin-1 (IL-1) and IL-2 mRNA in five human prostate cancer (PCa) cell lines and their effects on cellular proliferation and prostate-specific antigen (PSA) levels were examined. IL-1 was found in androgen-unresponsive PC-3 and DU-145 but not in the androgen-responsive LNCaP, MDA-PCA-2a and MDA-PCA-2b cell lines. IL-2 message was absent while that of GAPDH (positive control) was present in all five cell lines. IL-1 decreased while IL-2 increased PSA levels of near-confluent LNCaP cells after 24 h of treatment. IL-1 inhibited whereas IL-2 stimulated the growth of sub-confluent LNCaP cells (72 h). Neither cytokine affected the proliferation of DU-145 or PC-3 cells.     

Effect of PSK on Th1/Th2 balance in tumor-bearing mice

We investigated the effect of PSK on Th1/Th2 balance in tumor-bearing mice. PSK was intraperitoneally administered to Meth A-bearing BALB/c mice, and PSK caused regression of the Meth A tumor. The results of Winn assay suggested that the effect of PSK was dependent on CD4+T cells. Furthermore, spleen cells were cultured with mitomycin C-treated Meth A, after which the cytokine concentration was measured by ELISA. IFN-gamma production was increased and IL-4 showed almost no change in PSK-administered mice. In another experiment, PSK was orally administered to colon 26-bearing mice in which tumors were inoculated into the subserosal space of the cecum. Mesenteric lymph nodes cells were cultured with mitomycin C-treated colon 26 cells. IFN-gamma production was increased, but not so much as to be statistically significant, and IL-4 was significantly decreased in PSK-administered mice. PSK increased IFN-gamma and IL-12 p70 production and decreased IL-4 production when spleen cells were stimulated with Con A together with PSK in vitro. As suggested from these results, PSK might induce cytokine production that works for Th1 differentiation, and suppress cytokine production that works for Th2 differentiation, and shift the Th1/Th2 balance toward Th1 dominance in tumor-bearing mice.     

Enhancement of antiproliferative effects of interleukin-1beta and tumor necrosis factor-alpha on human prostate cancer LNCaP cells by coculture with normal fibroblasts through secreted interleukin-6.

The cell-cell interactions between tumor cells and stromal cells are considered to be important in the regulation of tumor development at primary and metastatic secondary sites. We studied the effects of various cytokines on the cell-cell interactions between androgen-dependent LNCaP or androgen-independent PC-3 human prostate cancer cell lines and normal fibroblasts using a co-culture system. Among the tested combinations of cytokines and fibroblasts, strong modulations of cytokine actions were seen in coculture with human normal fibroblasts WI-38. While interleukin (IL)-1beta or tumor necrosis factor-alpha (TNF-alpha) partially suppressed LNCaP cell growth in monoculture, each cytokine completely inhibited it in the case of coculture with WI-38 cells. On the other hand, they did not inhibit PC-3 cell growth significantly, regardless of monoculture or coculture. Conditioned medium prepared from WI-38 cells pretreated with IL-1beta or TNF-alpha also strongly inhibited LNCaP cell growth. In the conditioned medium, marked IL-6 secretion was induced from WI-38 cells by IL-1beta or TNF-alpha. Furthermore, neutralizing antibodies to IL-6 or IL-6 receptor abrogated the antiproliferative effects of IL-1beta- and TNF-alpha-pretreated WI-38 conditioned medium. These results demonstrate that the antiproliferative effects of IL-1beta and TNF-alpha on prostate cancer cells are enhanced by coculture with normal fibroblasts through some diffusible factor(s), such as IL-6, from the stimulated fibroblasts.     

Thalidomide up-regulates prostate-specific antigen secretion from LNCaP cells

Thalidomide has been shown to have species- and metabolic-dependent antiangiogenic activity in vitro and in vivo, suggesting its potential in treating human angiogenesis-dependent pathologies such as solid tumors. Based on promising preclinical studies, thalidomide has entered phase II clinical trials for prostate, brain, breast cancer, and Kaposi's sarcoma. However, the antiangiogenic mechanism of action is largely unresolved, as are its effects on tumor-associated gene expression, cytokine secretion, etc. We have investigated the effects of thalidomide on: 1) the secretion of prostate-specific antigen (PSA) in a human androgen-dependent prostate cell line; 2) growth and viability of human prostate cells; and 3) differential gene expression profiles of thalidomide-treated vs untreated human prostate cells. A human androgen-dependent prostate carcinoma cell line (LNCaP) and a human androgen-independent prostate carcinoma cell line (PC-3) were incubated with thalidomide 0.6, 6, or 60 7g/mL for 5-6 days. Secreted PSA from LNCaP cells was measured using a commercial enzyme-linked immunosorbant assay. Cell viability studies were conducted in both LNCaP and PC-3 cells using the same thalidomide concentrations. Furthermore, the differential gene expression of thalidomide-treated LNCaP cells was compared to that of untreated control cells using a commercially available human cancer cDNA expression array system. Thalidomide-treated LNCaP cells demonstrated increased PSA/cell levels at all concentrations tested compared to untreated control cells. Thalidomide demonstrated a cytostatic effect in LNCaP cells but had no appreciable effect on PC-3 cell viability compared to untreated control cells. Comparison of cDNA expression arrays hybridized with thalidomide-treated LNCaP cDNA probes suggests that thalidomide may up- or downregulate expression of angiogenesis-related genes, i.e., vitronectin, but these differential effects require further verification. Thalidomide over a range of doses has demonstrated nontoxic, cytostatic activity in LNCaP cells and significant upregulation of LNCaP cell PSA secretion in vitro. Furthermore, preliminary data from cDNA nucleic acid arrays of thalidomide-treated LNCaP cells suggest that thalidomide upregulates a potential angiogenic modulatory protein, the vitronectin precursor, which may eventually link thalidomide's antiangiogenic activity with modulation of angiogenic vascular integrin pathways.     

Effect of transfected interleukin-6 in non-tumorigenic and tumorigenic rat urothelial cell lines

It has been suggested that interleukin (IL)-6 plays a significant role in bladder carcinomas. We conducted the present experiment to determine whether over-expressed IL-6 enhanced the tumorigenicity of a weakly tumorigenic rat bladder carcinoma, and whether it was sufficient to induce a tumorigenic phenotype in a non-tumorigenic, anchorage-dependent rat urothelial cell line, MYP3. P3M6-10 and P3M6-12 (anchorage-independent but non-tumorigenic) and MYP3T6 (anchorage-independent and tumorigenic) were isolated from MYP3 after treatment with MNU in vitro. None of these clones produced IL-6. The cells were transfected with an expression vector containing human IL-6 cDNA. The transfectants secreted a large amount of IL-6. In MYP3T6t over-expression of IL-6 enhanced tumorigenicity markedly in nude mice, as evidenced by acceleration of the growth rate in vivo as well as of anchorage-dependent and -independent growth. In P3M6-10 and P3M6-12, the introduced IL-6 cDNA markedly enhanced their growth potential, both on a plastic surface and in soft agar, but did not induce a tumorigenic potential. In MYP3, introduced IL-6 weakly enhanced their growth on a plastic surface, but caused neither tumorigenesis in nude mice nor colony formation in soft agar. Expression of gpl30, an IL-6 signal transducer, was increased in IL-6-expressing clones, especially in the P3M6-10 and MYP3T6. We conclude that acquisition of the ability to synthesize endogenous IL-6 markedly accelerates the growth rate of weakly tumorigenic rat urothelial cells, but is not suffficient to induce a tumorigenic phenotype in non-tumorigenic cells. © 1996 Wiley-Liss, Inc.     

Tumor necrosis factor-alpha and interleukin-1 alpha production in cachectic, tumor-bearing mice

Weight-stable mice bearing a syngeneic, methylcholanthrene-induced, rapidly growing tumor lost approximately 22% of their lean tissue, became significantly hypoalbuminemic and had a marked increase in serum amyloid P concentrations during progressive tumor growth. Tumors from cachectic mice were producing both TNF-alpha and IL-1 alpha in vivo as documented by the presence of TNF-alpha and IL-1 alpha mRNA and immune-reactive protein for IL-1 alpha. Only spleens from tumor-bearing mice had statistically significantly elevated quantities of IL-1 mRNA. In general, alterations in tissue concentrations of IL-1 mRNA in tumor-bearing animals agreed qualitatively with those found in endotoxin-stimulated, non-tumor-bearing control mice. However, endotoxin-stimulated mice had significantly elevated tissue concentrations of TNF mRNA in spleen and livers, while TNF mRNA levels were not significantly increased in any host tissues. Cytokine mRNA levels in tumor tissue were not higher than those found constitutively in various tissues from non-tumor-bearing control animals. Plasma from tumor-bearing mice and endotoxin-stimulated controls contained high levels of IL-6 but low (endotoxin-stim.) or no measurable levels (tumor-bearing) of either IL-1 or TNF. When tumor cells from cachectic mice were placed into long-term cell culture, immune reactive TNF-alpha and IL-1 alpha were produced, but IL-6 bioactivity ws not produced in measurable amounts.     

Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency

Interleukin-6 (IL-6) is a pleiotropic cytokine that is capable of modulating the diverse functions of cells such as acute phase responses and inflammation. Excessive or insufficient production of IL-6 may contribute to certain diseases of the skin. The aim of this study was to investigate the possible role of IL-6 in the tumorigenesis of basal cell carcinoma (BCC). Initially, we transfected IL-6 expression vector, under the control of a CMV promoter, into human BCC cells and successfully obtained IL-6-overexpressing clones (BCC/IL-6-c1 and BCC/IL-6-c2) and a mixture (BCC/IL-6). DNA synthesis assay determined using (3)H-thymidine pulse incorporation revealed that IL-6-expressing BCC cells exhibited a much higher DNA synthesis rate than the neo control or parental BCC cells. We also detected a greater abundance of IL-6-expressing cell colonies formed in soft agar than in the vector control cells. Furthermore, BCC/IL-6 cells, but not vector control cells, were resistant to UV and photodynamic therapy (PDT)-induced apoptosis, as confirmed using DNA fragmentation and morphologic change analyses. Immunoblot analysis showed that Mcl-1, an anti-apoptotic protein, was specifically up-regulated IL-6 transfectants but not in the control cells. Transient transfection of IL-6 transfectants with antisense mcl-1 greatly enhanced their apoptosis frequency by UV treatment. In tumorigenesis assay, IL-6 transfected clones formed tumors in nude mice more rapidly than the control cells. These tumors appeared to be highly vascularized using pathological examination. Supportive of this finding, we found that IL-6 transfected cells expressed elevated levels of two angiogenic factors, cyclooxygenase (Cox)-2 and vascular endothelial growth factor (VEGF). These results suggest that overexpression of IL-6 enhances the tumorigenic activity of BCC cells by both suppressing apoptosis and actively promoting angiogenesis.     

Rapid reversal of interleukin-6-dependent epithelial invasion in a mouse model of microbially induced colon carcinoma

Chronic inflammation of mucosal surfaces renders them increasingly susceptible to epithelial cancers both in humans and mice. We have previously shown that anti-inflammatory CD4(+)CD45RB(lo)CD25(+) regulatory (Treg or T(R)) lymphocytes down-regulate inflammation and block development of bacteria-triggered colitis and colorectal cancer (CRC) in 129/SvEv Rag2-/- mice. Interestingly, T(R) cells collected from Interleukin (IL)-10-deficient cell donors not only failed to suppress carcinogenesis but instead promoted invasive mucinous colonic carcinoma with a strong gender bias expressing in male mice. We found we show that peritoneal invasion in this model is dependent on pleiotropic cytokine IL-6. Mucinous carcinoma arose rapidly and consistently after treatment with IL10-/- T(R) cells, which were found to express Foxp3+ and localize throughout tumor tissue. Carcinogenesis was rapidly reversible with transfer of wild type IL10-competent T(R) cells. Likewise, treatment with IL10-Ig fusion protein was sufficient to revert the lesions histologically, and restore inflammatory cytokine and oncogene expression to base line levels. These studies indicate an essential role for IL 6 in this CRC phenotype. Furthermore, immune-competent T(R) cells were important not only for preventing pathology but also for constructive remodeling of bowel following tumorigenic microbial insults. These data provide insights into etiopathogenesis of inflammation-associated epithelial invasion and maintenance of epithelial homeostasis.     

Mice vaccination with interleukin 12-transduced colon cancer cells potentiates rejection of syngeneic non-organ-related tumor cells

Cell-based gene therapy after cytokine gene transfer is being investigated for autologous and allogeneic vaccination in cancer therapy. Here we show that mice vaccinated with 3-5 x 10(6) interleukin 12 (IL-12) gene-transduced CT26 colon cancer cells developed a long-lasting antitumor immune memory able to reject not only parental cells but also syngeneic, LM3 mammary, and MCE fibrosarcoma tumorigenic cells. In contrast, mice vaccinated with 0.5-1 x 10(6) CT26 cells transduced with pBabe neo IL-12 retrovirus cells (CT26-IL12) were only able to reject parental cells. An increase in the total circulating levels of IgG2a and a clear shift toward a systemic Th1 response developed, regardless of the amount of injected CT26-IL12 cells. On the contrary, a strong increase in anti-CT26-specific IgG2a levels was observed only when 3-5 x 10(6) CT26-IL12 cells were injected. Immunocompetent mice vaccinated with 3-5 x 10(6) CT26-IL12 cells developed local nodules for a few days, which then ceased growing. These nodules comprised mainly blood vessels, suggesting that an angiogenic process was taking place. CD8+ T cells were responsible for the anti-LM3 tumor cell memory, whereas CD4+ T cells were not involved. Splenocytes and lymphocytes obtained from mice immunized against CT26 cells were able to kill LM3 cells in vitro. Adoptive transfer of lymphocytes obtained from animals immunized against CT26 colon cancer cells suppressed LM3 mammary tumor growth in tumor-bearing mice. The present studies raised the possibility of isolating CTL clones and identifying CTL epitopes shared by different tumor cell types, which can be a target for cancer therapy.