Improved Therapeutic Effectiveness By Combining Recombinant CXC Chemokine Ligand 10 With Cisplatin In Solid Tumors

A common devastating disease is malignant tumor, which causing great insult to the health and the life quality of human. Cytotoxic chemotherapy is the mainstay of medical approaches to treat life-threatening cancer, although several novel strategies or drugs are under active research. Cisplatin remains the most widely used the first line element of cytotoxic chemotherapy to solid tumors. Complete remission and long survival had been observed in some cases. Nevertheless, the efficacy of cisplatin based on the treatment is limited in curing most tumors due to relapse, and a large proportional of patients still being succumbed even if under continued treatment. The recent findings from other groups indicated that in order to successfully counteract tumor progression, the traditional anti-tumor regime and strategy, such as chemotherapy or radiotherapy should be employed in combination with agents thwarting new vasculature formation.CXC chemokine ligand 10 (CXCL10), also known as y interferon inducible protein 10 (IP-10), is firstly found by interferon y or lipopolysaccharide stimulated monocytes, endothelial cells, and keratinocytes. It has also been reported to be a potent inhibitor of angiogenesis, besides displaying a potent thymus-dependent anti-tumor effect. Therapy withrecombinant CXCL10 or with virus/non-virus vector expressing CXCL10 is effective in reducing the rate of tumor growth, while it fails to induce tumor complete regression, which suggests that the further treatment may require supplemental drugs targeting directly the tumor cells. To date, the combination of CXCL10 with cisplatin remains to be explored. Based on different mechanism of the two agents, we hypothesized that treatment of CXCL10 in combination with cisplatin would improve the survival in tumor bearing immunocompetent mice.MethodsThe nucleotide sequence encoding CXCLIO cDNA (Accession No. human x02530 and murine m33266) was polymerase chain reaction-amplified from pBLAST-IP-10 (InvivoGen, San Diego, CA) and cloned into an expression plasmid pET-32a (Novagen, Madison, WI). Purification of CXCL10 was conducted in the AKTA purification system. Recombinant CXCL10 protein after enterokinase digestion were collected and refolded. The chemotaxis assay of CXCL10 to lymphocytes indicates that a protein with biological activity has been obtained.CT26 colon adenocarcinoma (CT26) and Lewis lung cancer (LL/2) models were established in the immunocompetent BALB/c and C57BL/6mice, respectively. Animals were randomly subdivided into four groups and treated with PBS, CXCL10, DDP or CXCL10 + DDP, respectively. Administration were done with either CXCLIO s.c. at 25 μg/kg/day once daily for 30 days, cisplatin cycled twice (5 mg/kg i.p. on days 14 and 21 after the initiation of CXCL10), or both agents together. Tumor volume and survival were observed.The alginate beads and CD31 immunohistochemical are done to assay the inhibition of angiogenesis. TUNEL is used to determine the apoptosis in tumor tissues. ResultsRecombinant -plasmids cloned into the pET32 vector identified by the enzyme digestion before further verifiing in DNA sequence. Results showed that the human and murine CXCL10, with proper reading frame, are completely in line with the sequences of GenBank. Recombinant human and murine CXCL10 protein was expressed in E. coli BL21(DE3), under 1.0 mmol/L isopropyl-b-D-thiogalactopyranoside. Compared with before induction, a new protein band in tricine polyacrylamide gel electrophoresis (Tricine-PAGE) appeared in the expected site. Recombinant CXCL10 is mainly expressed in inclusion body. The protein are well washed under the condition of wash buffer containing 200 mmol/L immidazole (pH9.0) before electrphorsis and Western Blot. Chemotaxis assay indicated that refolding of CXCL10 resulted in a product with complete biological activity for further animal experiment.Recombinant CXCL10 clearlly inhibited the growth of tumor and prolonged the survival of tumor bearing mice as compared with PBS control (P<0.05). CXCL10 plus cisplatin reduced tumor growth in two tumor models more effectively, although cisplatin or CXCL10 individually resulted in suppression of tumor growth and improved survival time of tumor bearing mice. However, no completely remission had been found. Lung metastasis nodules were also counted under dissecting microscope after 4 days terminating treatment in LL/2 tumor model. Results demonstrated that the number of the matastatic nodules were decreased in combined treated group or CXCL10 agent alone relative to PBS control (P<0.05). Sections of HE staining also showed the apparent difference between CXCL10 and PBS group. A little reduction of metastatic nodules in DDP administered group was observed, although with no significant difference being found compared with PBS control (P=0.12).The vascular plexus of the tumor in PBS group formed a ladder-like pattern, which were richly morphology and irregular in the alginate experiment.Vascularization of alginate beads was apparently deminished, and FITC-dextran uptake was significantly decreased in CXCL10 treated mice as compared with that in PBS controls (p<0.05).The inhibition of angiogenesis was also determined by the immunohistochemistry with CD31 in tumors tissues embedded in paraffin. The combination of CXCL10 with DDP significantly reduced the number of vessels as compared with control groups, including PBS or DDP alone (P<0.05). No statistically significant difference has been obtained between DDP and PBS group (P>0.05).To explore the role of therapy on apoptosis of tumor cells, tumor resected were subjected to TUNEL assays for the determination of apoptotic index. DDP or CXCL10 alone treatment affected the apoptosis rate of tumor cells, whereas the density of apoptotic cancer cells elevated after the combined CXCL10 with DDP therapy (P<0.01). Histologically, the significant necrosis can also be gained in the combined treatment group. Control tumors displayed little or no tumor tissue necrosis and had normal capillaries surrounding nests of confluent tumor cells. Leukocytes infiltration (mainly including monocytes and CD8+ lymphocytes) exhibiting clustering-like aggregation was apparently found in the tissue of CXCL10 alone or CXCL10 in conjunction with DDP group. Little or no leukocytes had been found in PBS or DDP treated groups.No adverse consequences were showed in gross measures such as weight loss, ruffling of fur, life span, behavior, or feeding. No reactive antibody in the serum can be determined by ELISA. None of pathologic changes in liver, lung, kidney, etc. have been found by microscopic examination, besides mildly spleen proliferation in CXCL10 treated mice.The present study pave the basis for the future investigation of CXCL10. In addition, it may be of importance to the further exploration of the potential application of this combined approach in the treatment of lung and colon carcinoma. One could also achieve better antitumor response by combiningCXCL10 with other chemotherapies or radiation. Conclusions1. The recombinant expressing plasmid of human and murine CXCL10 has been constructed. CXCL10 protein has been expressed in E. coli BL21(DE3), under the induction of 1.0 mmol/L isopropyl-b-D-thiogalactopyranoside.2. After purification, enterokinase digestion and refolding, chemotactic assay indicates that CXCL10 with biological activity has been obtained.3. CXCL10 as an agent alone potentially inhibits the growth of tumors and prolongs the survival time of tumor bearing mice.4. CXCL10 plus cisplatin reduces tumor growth more effectively, although cisplatin or CXCL10 individually resultes in suppression of tumor growth and gained the survival time. To our knowledge, this is the first report of CXCL10 in conjunction with cisplatin in the thwarting to solid tumors.5. CXCL10 significantly inhibited angiogenesis as assessed by alginate model and CD31 immunohistochemical.6. Improved therapeutic efficacy by CXCL10 in combination with cisplatin also results from the increased rate of apoptosis and necrosis in tumor tissues, as well as the activation of host immune.7. No increased toxicity had been found in the combined group, which indicating that CXCL10 is a well-tolerated angiogenesis inhibitor.8. The present study pave the basis for the futher investigation of CXCL10. It also may be of importance to the further exploration of the potential application of this combined approach in the treatment of lung and colon carcinoma.