| Cancer is still a terrible illness to hurt people in all of the world. The development of drug resistance is a major problem encountered in cancer chemotherapy. Except the gene instability of cancer cells, the pathophysiological characteristics of the tumor are also the major reasons that contribute to the drug resistance. The pathophysiological characteristics of the tumor give rise to the abnormal organization, structure, and function of tumor blood vessels. These abnormalities contribute to heterogeneous blood flow, vascular permeability, and microenvironment. Each of these abnormalities forms a physiological barrier to the delivery of therapeutic agents to tumors. Furthermore, the metabolic microenvironment in tumors such as hypoxia and acidosis hinder the efficacy of anti-tumor treatments such as radiation therapy and chemotherapy.Jain proposed an additional rationale for anti-angiogenic therapy 'normalizing' tumor vasculature before its destruction. The transient normalization of tumor vessels produces a temporary increase in oxygen and nutrient delivery to the cancer cells that surround these "normalized" vessels. This might be expected to enhance the proliferation of these cells and hence to accelerate tumor growth. Conceivably, this therapy might also induce normalization and improve oxygenation and drug penetration into tumors. Indeed, blockade of VEGF signaling passively prunes the immature and leaky vessels of transplanted tumors in mice and actively remodels the remaining vasculature so that it more closely resembles the normal vasculature. These morphological changes are accompanied by functional changes-decreased interstitial fluid pressure, increased tumor oxygenation, and improved penetration of drugs in these tumors.Artemisinin, a sesquiter lactone isolated from the plant Artemisia annua L., and its derivatives are presently used in various countries as an antimalarial drug with little toxicity to human and have a pontent effect on chloroquine-resistant malarial parasites. Dihydroartemisinin is a more water-soluble and effective antimalarial analog of artemisinin. Recently, it was reported that artemisinin analogs also showed antitumor activity in vitro and in vivo. In our previous study, we have shown dihydroartemisinin inhibits the VEGF expression in solid tumor xenograft, K562 cells, RPMI8226 cells and exhibits the potent antiangiogenesis effect. However, to our knowledge, the enhancement of artemisinin analogs to chemotherapy drugs has not yet been demonstrated.In the present study, we observed the therapeutic effect of dihydroartemisinin combined with cyclophosphamide in the murine Lewis lung carcinoma in vivo. The VEGF expression in xenotransplanted carcinoma was tested by immunohistochemical stainings. Besides, we investigated the antiproliferation and inducing apoptosis effect of dihydroartemisinin on LLC cells, and assessed the expression of VEGF receptor KDR/flk-1 in LLC cells in vitro.Results1. Tumor volume, tumor weight and animal weightMurine Lewis lung carcinoma xenograft study showed dihydroartemisinin have the ability to enhance the chemotherapeutic effect of cyclophosphamide. In vivo, dihydroartemisinin combined with cyclophosphamide resulted in the significant regression of murine Lewis lung carcinoma transplanted tumors compared with either therapy alone. The combination was more effective when cyclophosphamide was administered 7 days after dihydroartemisinin compared with when cyclophosphamide was administered 5 days after dihydroartemisinin. Moreover, the incidence of spontaneous pulmonary metastasis was completely inhibited by the combination of dihydroartemisinin with cyclophosphamide. These studies indicate that dihydroartemisinin enhances the ability of cyclophosphamide to inhibit tumor growth and metastasis in murine Lewis lung carcinoma. Drug-induced body weight loss was not significantly different in any of the therapeutic groups.2. Expression of VEGF in tumor xenograftExpression of VEGF was confirmed by the presence of brown stained cytoplasm in LLC cells. Most LLC cells in the control group and cyclophosphamide treated group showed strong expression of VEGF, but the expression in dihydroartemisinin single treated groups and the combined treated groups were weaker. No significant immunohistochemical reaction occurred in the native control slips.3. The anti-proliferation effect of dihydroartemisinin in LLC cellsTreated with dihydroartemisinin at concentrations of more than 5μmol·L-1 for 48h inhibited the growth of LLC cells in a dose dependent manner as shown in MTT assay. Dihydroartemisinin at 5μmol·L-1, 10μmol·L-1, 20μmol·L-1, 40μmol·L-1, 80μmol·L-1, 160μmol·L-1 inhibited the growth of cells by8.74%, 15.05%, 29.24%, 45.78%, 62.37%, 70.53%respectively. The IC50 value of dihydroartemisinin for growth inhibition of LLC cells was 53.14μmol·L-1, and 95% confidence interval was 48.69-58.18μmol·L-1.4. Morphological changes of apoptosis in LLC cellsAcridine orange/ethidium bromide was used to assess changes in nuclear morpholory following dihydroartemisinin treatment. The nuclei in normal cells were normal and exhibited diffused staining of the of the chromatin. However, after exposure to 40, 80μmol·L-1 dihydroartemisinin for 48h, LLC cells underwent typical morphologic changes of apoptosis such as chromatin condensation and shrunken nucleis. Therefore, these morphological changes suggested the occurrence of apoptosis in LLC cells after treated with dihydroartemisinin.5. Percentage of cells undergoing apoptosisMoreover, the morphological alterations of DNA in dihydroartemisinin treated LLC cells were supported by flow cytometry analysis. The dose-related increase in dihydroartemisinin-treated LLC cells apoptosis initially analyzed using flow cytometry. After cells were treated with 20, 40, 80μmol·L-1 dihydroartemisinin for 48h, the rate of apoptosis was detected by PI-flowcytometry. The percentage of apoptosis cells increase to 5.54%, 9.92%, 13.86%, respectively.6. Expression of KDR/flk-1 in LLC cellsWestern blot was exploited to detected the expression of KDR/flk-1 in LLC cells affected by dihydroartemisinin. We treated LLC cells in vitro with various concentration of dihydroartemisinin, and found that increasing concentration of dihydroartemisinin lead to a stepwise reduction in KDR/flk-1 expression. Compared with vehicle control, the levels of KDR/flk-1 in LLC cells were decreased by 33.8%, and 12.6% after treated with 40, 80μmol·L-1 dihydroartemisinin for 48h, respectively(p<0.001).Statistical analysisAll experiments were performed in triplicate unless otherwise noted, results were expressed as mean±standard deviation.. The mean differences between samples were evaluated by t test using software SPSS version 10.0. Throughout the work, p values less than 0.05 were regarded to be statistical significant.DiscussionMuch of the vasculature in tumors is disorganized, leaky, and structurally abnormal. The structural eccentricities include absent, reduced, or altered basement membranes and periendothelial support cells such as pericytes; excessively dilated or constricted vessels; and corkscrew-like tortuosities. Tumor vessel leakiness can lead to extravasation of plasma proteins and fluid, producing high interstitial fluid pressures (IFPs) within tumors, which in turn may impede the delivery and diffusion of certain cancer drugs, especially large molecules or immune killer cells. In addition, the flow and perfusion of blood in many of the tumor vessels may be impaired, which, as noted above, can produce localized and transient areas of hypoxia. As a result, nearby tumor cells may enter a quiescent state that would reduce their inherent sensitivity to chemotherapy and radiation.Jain proposed an additional rationale for anti-angiogenic therapy: 'normalizing' tumor vasculature before its destruction to improve the delivery of drugs and oxygen. VEGF is overexpressed in the majority of solid tumors. Thus, if one were to judiciously down-regulate VEGF signaling in tumors, then the vasculature might revert back to a more "normal" state. Indeed, blockade of VEGF signaling passively prunes the immature and leaky vessels of transplanted tumors in mice and actively remodels the remaining vasculature so that it more closely resembles the normal vasculature. This "normalized" vasculature is characterized by less leaky, less dilated, and less tortuous vessels with a more normal basement membrane and greater . coverage by pericytes. These morphological changes are accompanied by functional changes—decreased interstitial fluid pressure, increased tumor oxygenation, and improved penetration of drugs in these tumors.In the present study, we demonstrated that dihydroartemisinin enhance the therapeutic effects of cyclophosphamide by increasing its inhibitory effect on tumor growth, metastasis. The combined drugs had an additive effect on tumor growth inhibition. We examined the effect of potentiation of cyclophosphamide antitumor effects in murine lung tumor xenografts by effects of the angiogenesis inhibitor dihydroartemisinin: on tumor neovascularization. Murine Lewis lung carcinoma xenograft study showed dihydroartemisinin have the ability to enhance the chemotherapeutic effect of cyclophosphamide. In vivo, dihydroartemisinin combined with cyclophosphamide resulted in the significant regression of murine Lewis lung carcinoma transplanted tumors compared with either therapy alone. The combination was more effective when cyclophosphamide was administered 7 days after dihydroartemisinin compared with when cyclophosphamide was administered 5 days after dihydroartemisinin. Moreover, the incidence of spontaneous pulmonary metastasis was completely inhibited by the combination of dihydroartemisinin with cyclophosphamide. These studies indicate that dihydroartemisinin enhances the ability of cyclophosphamide to inhibit tumor growth and metastasis in murine Lewis lung carcinoma. Drug-induced body weight loss was not significantly different in any of the therapeutic groups.Although the mechanism of dihydroartemisinin function as an inhibitor of angiogenesis has been well studied, the mechanism by which dihydroartemisinin enhanced the therapeutic effect of cyclophosphamide have been less well characterized. In the present study, we assessed the effect of dihydroartemisinin combined with cyclophosphamide on VEGF and VEGF receptor expression in murine LLC cells. We first investigated the anti-proliferation effect of dihydroartemisinin in LLC cells in vitro. The result indicated that dihydroartemisinin could effectively inhibit the proliferation of LLC cells, and the inhibitory effect of dihydroartemisinin on the proliferation of LLC cells was in a dose dependent manner. Artemisinin derivates in range of 20-180μmol·L-1 have been reported to inhibit cancer cell proliferation in vitro. Our study also showed that the IC50 value of dihydroartemisinin on LLC cells is 53.14μmol·L-1. In order to analyse the effect of dihydroartemisinin on VEGF and VEGF receptor KDR/flk-1 expression in LLC cell, we assessed the level of VEGF expression by immunohistochemistry in vivo and the VEGF receptor KDR/flk-1 expression by western blot in vitro. All these experiments suggested that dihydroartemisinin could inhibit the expression VEGF and VEGF receptor effectively in LLC cells.Previous reports have also demonstrated that dihydroartemisinin inhibits the growth of HUVEC, downregulated the. expression VEGF and VEGF receptor in HUVEC, by which dihydroartemisinin have an inhibitory effect on angiogenesis. The downregulated of VEGF and VEGF receptor in LLC cells may play an important role in the normalization of tumor vessel. We think this may one mechanism of dihydroartemisinin enhance the therapeutic effect of cyclophosphamide.In addition to the antiproliferation effect of dihydroartemisinin LLC cells, dihydroartemisinin was also found to induce LLC cells apoptosis from our data. This was consistent with previous reports which indicate that the antitumor effect of dihydroartemisinin was ascribe to the rapid induction of apoptosis in cancer cells after treated with dihydroartemisinin. It has been showed that elevated circulating VEGF level will confer VEGFR+ expression tumor cells with great survival potential and resistance to apoptosis in an autocrine fashion. In our study, we confirmed that dihydroartemisinin downregulated the expression of VEGF receptor KDR/flk-1 in LLC cells, and induce LLC cells apoptosis.The recent finding of VEGFR expression on tumor cells may in part explain the improved efficacy of anti-VEGF treatment in combination with cytotoxic therapy. To date, there are no experimental data directly addressing this hypothesis. In theory, however, inhibition of VEGFR signaling in tumor cells by anti-VEGF/VEGFR therapy may potentiate the effects of cytotoxic drugs by inhibiting antiapoptotic regulators, or some other survival mechanisms in tumor cells. In summary, the present study provides evidence that dihydroartemisinin enhances the therapeutic effects of cyclophosphamide by increasing its inhibitory effect on tumor growth, metastasis in murine Lewis lung carcinoma. Induction of apoptosis by dihydroartemisinin and the downregulattion of the expression of VEGF receptor KDR/flk-1 in LLC cells would appear to work synergistically together to enhance efficacy. These studies indicate that dihydroartemisinin and cyclophosphamide have synergistic effect, providing a clear rationale for investigation in future clinical trials. |
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