重组沙门氏菌VNP20009-M对骨肉瘤的治疗作用

目的 研究携带甲硫氨酸酶基因的减毒沙门氏菌VNP20009-M对骨肉瘤的治疗作用及其机制。方法 将重组沙门氏菌VNP20009-M与骨肉瘤细胞MNNG-HOS共培养;骨肉瘤细胞MNNGHOS、U2OS及SaoS-2均高表达甲硫氨酸酶基因后探索细胞增殖、迁移及凋亡能力的变化;构建MNNG-HOS裸鼠皮下荷瘤模型,评价不同剂量的VNP20009-M在动物模型中的治疗效果。结果 通过质粒PCR验证甲硫氨酸酶基因只存在于目的菌株VNP20009-M中,且具有较高的甲硫氨酸酶活性,重组沙门氏菌构建成功。VNP20009-M显著诱导骨肉瘤细胞MNNG-HOS的凋亡。相比对照组,甲硫氨酸酶基因过表达的骨肉瘤细胞增殖、迁移能力被显著抑制。VNP20009-M治疗后小鼠皮下肿瘤生长被显著抑制。结论 VNP20009-M可诱导骨肉瘤细胞凋亡并抑制癌细胞增殖和迁移,可以作为一种新型高效的药物为临床提供新的治疗方式。     

Antitumor effect of actinidia chinensis polysaccharide on murine tumor

A new polysaccharide compound (ACPS-R) has recently been isolated from the root of Actinidia Chinensis Planch. When given intraperitoneally to the transplantable tumor bearing mice at dose of 75-125 mg/kg, the tumor inhibition rate was more than 88.8% in Ehrilich ascitic cancer (EAC) or ascitic form of hepatoma (HepA) and more than 49.6% in solid hepatoma (HepS). The treatment effect of ACPS-R on EAC at dose of 15 mg/kg and 22.5 mg/kg, respectively. ACPS-R could also prolong the life of EAC-or P388-bearing mice, and increase the percentage of EAC-free mice. In addition, when ACPS-R was used in combination with 5-Fu, the antitumor effect was enhanced as compared with 5-Fu alone. A marked increase in cAMP levels and cAMP/cGMP ratio of spleen of EAC-bearing mice were observed after treatment of ACPS-R. The increase of both parameters nearly reached the normal levels of healthy mice. The increases of cAMP, cAMP/cGMP and tumor remission had statistical significance. It showed an intermediate inhibitory effect of ACPS-R on DNA synthesis by incorporating 3H-TdR into EAC cells. The results indicated that ACPS-R acts as a new antitumor polysaccharide, and the treatment effect of Actinidia root in folk medicine is probably related to ACPS-R.     

Oral cytokine gene therapy against murine tumor using attenuated Salmonella typhimurium.

An attenuated strain of Salmonella typhimurium was used as a vehicle for oral gene therapy against murine tumor. Eukaryotic expression vectors containing genes of human interleukin-12 (hIL-12), human granulocyte/macrophage colony-stimulating factor (hGM-CSF), mouse (m)IL-12, mGM-CSF and green fluorescent protein (GFP) were used to transform attenuated Salmonella (SL3261), and such transformants were administered orally to BALB/c and C57BL/6 mice. As a reporter gene, GFP expression in murine liver, spleen, tumor, intestine and kidney was confirmed by confocal and flow cytometry. Soluble cytokines were detected in murine sera, and the concentrations were much higher than those of the control, which contributed to the increased number of cytotoxic T cells and prolongation of survival. Oral cytokine gene therapy using live attenuated Salmonella demonstrated a significant protection against the development of two unrelated murine tumors. These results suggest that such gene therapy has the potential to be simple, effective and (above all) safe against tumor.     

Oral delivery of tumor-targeting Salmonella for cancer therapy in murine tumor models

Tumor-targeting bacteria have been investigated intensively in recent years as anticancer agents. To ensure the reliability of infection, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. However, systemic infection of bacteria is rather inconvenient and carries the risk of obvious toxicity. Here we tested whether Salmonella typhimurium VNP20009, a tumor-targeting strain, could be administrated orally for tumor therapy. Tumor-targeting potential, antitumor effects, as well as toxicity of orally administrated VNP20009 were investigated in this study. Oral delivery of VNP20009 not only exhibited high tumor-targeting potential, but also led to a significant anticancer effect by delaying tumor growth and prolonging survival in murine tumor models. As part of combination therapy, orally administrated bacteria notably improved the antitumor effect of cyclophosphamide. In vitro and in vivo studies showed that VNP20009 significantly induced tumor cell apoptosis. No obvious toxicity was observed during the treatments with oral inoculation of VNP20009. Comparative analysis of toxicity in tumor-bearing and tumor-free mice further revealed that orally administrated Salmonella had high safety compared to conventional systemic infection of bacteria. The findings indicated that oral administration of tumor-targeting bacteria is effective and safe. This approach provides a novel avenue in the application of bacteria as a potential antitumor agent.     

Anti-tumor efficacy of Cloretazine (VNP40101M) alone and in combination with fludarabine in murine tumor and human xenograft tumor models

Cloretazine (VNP40101M), a new sulfonylhydrazine alkylating agent, has demonstrated broad-spectrum anti-tumor activity in preclinical studies. In this study, Cloretazine was evaluated both as a monotherapy and in combination with fludarabine in murine tumor and human tumor xenograft models. Cloretazine significantly inhibited the growth of subcutaneously implanted tumors, including B16F10 murine melanoma in C57BL/6 mice, and H460 human lung carcinoma and WiDr human colon carcinoma in athymic nude CD1 mice. The inhibition of tumor growth by Cloretazine was dose dependent, increasing from 42.2 to 87% as the dose escalated from 100 to 150 mg/kg. Cloretazine showed equivalent efficacy but lower toxicity compared to cyclophosphamide in these models. The combination therapy, consisting of a single dose of 10 mg/kg Cloretazine plus five doses of 70 mg/kg fludarabine, given every other day intraperitoneally, significantly increased the long-term survival of BDF1 mice bearing the L1210 murine leukemia. On Day 65 post-tumor implantation, the combination therapy yielded a 90% survival rate compared to 40% for Cloretazine alone and 0% for fludarabine alone.     

Comparison of the selective targeting efficacy of Salmonella typhimurium A1-R and VNP20009 on the Lewis lung carcinoma in nude mice

Salmonella typhimurium A1-R is auxotrophic for arg and leu, which attenuates growth in normal tissue but allows high tumor targeting and virulence. A1-R is effective against metastatic human prostate, breast, and pancreatic cancer as well as osteosarcoma, fibrosarcoma, and glioma in clinically-relevant mouse models. VNP20009 is also a genetically-modified strain of Salmonella typhimurium that has been tested in Phase I clinical trials, but is more attenuated than S. typhimurium A1-R and in addition of multiple amino-acid auxotrophs, is purine auxotropic with the purI mutation. In the present study, mouse Lewis lung carcinoma-bearing nude mouse models were treated with S. typhimurium A1-R or VNP20009. S. typhimurium A1-R and VNP20009 were both eliminated from the liver and spleen approximately 3-5 days after administration via the tail vein. However, A1-R showed higher tumor targeting and inhibited the Lewis lung carcinoma to a greater extent than VNP20009, with less body weight loss. The mice tolerated S. typhimurium A1-R to at a least 2-fold higher dose than VNP20009 when the bacteria were administered iv. The results of the present study suggest that S. typhimurium A1-R has greater clinical potential than VNP20009.     

Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models

Antitumor activity of UCN-01 (7-hydroxy staurosporine), a selective inhibitor of Ca2+- and phospholipid-dependent protein kinase C, was examined in comparison with staurosporine, a nonselective inhibitor of protein kinases, on human and murine tumor cell lines which have some aberrations in cellular signal transduction. UCN-01 inhibited the growth of five tumor cell lines about 9 to 90 times less potently than staurosporine in vitro. UCN-01 showed an in vivo antitumor effect against three human tumor xenografts [epidermoid carcinoma A431 (c-erbB-1 overexpression), fibrosarcoma HT1080 (N-ras activation), and acute myeloid leukemia HL-60 (N-ras activation)], giving a minimum treated/control ratio of 0.40 (P less than 0.01), 0.17 (P less than 0.01), and 0.61 (P less than 0.05), respectively. UCN-01 also exhibited significant antitumor activity against two murine tumor models (fibrosarcoma, K-BALB and M-MSV-BALB), which activated the v-ras and v-mos oncogenes, showing a minimum treated/control ratio of 0.27 (P less than 0.01) and 0.21 (P less than 0.01). Staurosporine did not show significant antitumor activity against any of these five tumors. UCN-01 inhibited the down-modulation of epidermal growth factor receptor caused by phorbol 12-myristate 13-acetate in A431 cells at a near 50% inhibitory concentration for cell growth. These results imply that UCN-01 is a promising antitumor agent which has a novel mechanism(s) of action.     

Antitumor activity of MST-16, a novel derivative of bis(2,6-dioxopiperazine), in murine tumor models

Summary We studied the antitumor activity of newly synthesized bis(1-acyloxymethyl) derivatives of 4,4′-(1,2-ethanediyl)bis(2,6-piperazinedione) using i.p.-i.p. models of P388 leukemia and B16 melanoma. As a result, we found 4,4′-(1,2-ethanediyl)bis(1-isobutoxycarbonyloxymethyl-2,6-piperazinedione) (MST-16) to possess considerable therapeutic activity. MST-16 showed not only marked life-prolonging effects in both P388 leukemia- and B16 melanoma-bearing mice but also a greater therapeutic ratio than did its parent compounds, ICRF-154 and ICRF-159. Further studies revealed that MST-16 has considerable therapeutic activity against a number of other tumors such as ascitic forms of L1210 leukemia, colon 26 adenocarcinoma, and MH-134 hepatoma and solid forms of B16 melanoma, Lewis lung carcinoma, colon 38 adenocarcinoma, and M5076 fibrosarcoma. These results suggest that MST-16 is very promising as an antitumor agent.     

Antitumor effect of arsenic trioxide in murine xenograft model

Arsenic trioxide, As₂O₃ (ATO), has been established to be an effective agent for treating acute promyelocytic leukemia, but its effect on solid tumors has not been fully explored. In the present study in a murine xenograft system, we found that ATO significantly inhibited tumor growth of the inoculated human hepatocellular carcinoma cell line HuH7 when administered either intravenously or intratumorally. Pathological examination revealed that ATO induced extensive cell death in the tumor. Some of the dead cells in intratumorally ATO-treated mice showed characteristic features of apoptosis, such as nuclear condensation and fragmentation, and were TUNEL-positive. The measurement of arsenic by using particle induced X-ray emission revealed that arsenic was accumulated more in the tumor than in brain, kidney or liver after the intravenous injection of ATO, which is consistent with the hemorrhagic cell death observed in ATO-treated tumor tissues. Thus, ATO appears to have potential for the treatment of solid tumors, as well as hematopoietic malignancies.     

Effects of disodium etidronate in murine tumor models

This study was designed to elucidate the effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) in experimental rodent tumors. EHDP had no antitumor activity against the L1210 leukemia implanted i.p. and against sarcoma 180, Lewis lung carcinoma (3LL) and Walker 256/B carcinoma injected i.p., s.c. or i.m. respectively. EHDP did not interfere with the antitumor activity of commonly used conventional chemotherapeutic agents (adriamycin, cyclophosphamide, 5-fluorouracil, bis-chloroethylnitrosourea) in the L1210 and 3LL models. EHDP reduced proportionally to the dose the hypercalcemia and hypercalciuria due to the Walker 256/B carcinoma growth. In an effort to evaluate whether EHDP-treated osseous tissues were more refractory to tumor growth, cells from sarcoma 180 and 3LL carcinoma were implanted intratibially (i.t.). Growth of 3LL cells was not consistently affected by EHDP, whereas a modest, but significant, growth inhibition was consistently observed with sarcoma 180 injected i.t. Growth of sarcoma 180 implanted i.p. or s.c. was not reduced by this drug, thus suggesting that inhibition of i.t. sarcoma 180 was in fact related to alterations of osseous tissues by EHDP. Inoculation of Walker 256/B carcinoma intra-aortically resulted in osteolytic bone lesions in the hind limbs. EHDP inhibited the formation of bone metastasis under these conditions.     

Antitumor activity of amidoximes (hydroxyurea analogs) in murine tumor systems

A series of amidoximes was prepared and evaluated for possible antitumor activity against L1210 leukemia. Three of the most active compounds in the L1210 system, formamidoxime, acetamidoxime, and 2-aminoacetamidoxime hydrochloride, were also active against P388 leukemia. Acetamidoxime was marginally active against Lewis lung carcinoma. The active amidoximes showed best activity against L1210 leukemia when given two times daily, 5 hours apart, for 8 days. Most of the amidoximes produced excessive central nervous system stimulation.     

Antitumor effect of Sarcophaga lectin on murine transplanted tumors

Sarcophaga lectin, a lectin purified from the hemolymph of Sarcophaga peregrina (flesh fly) larvae, was found to be therapeutically effective against both ascitic and solid tumors. It was especially effective when injected directly into or around nodules of the syngeneic tumor Meth A transplanted intradermally into BALB/c mice. It also induced cytotoxic activity against L-929 cells when it was added to the culture medium of macrophages in vitro. Similar cytotoxic activity was detected transiently in the serum when it was injected into the abdominal cavity of mice, in which sarcoma 180 cells had been inoculated. Probably, this cytotoxic activity induced by Sarcophaga lectin partly contributes to the elimination of tumors. The possible role of this cytotoxic activity is discussed from the viewpoint of ontogeny.     

Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model

Some anaerobic and facultative anaerobic bacteria have been used experimentally as anticancer agents because of their selective growth in the hypoxia regions of solid tumors after systemic administration. We have previously shown the feasibility of using attenuated Salmonella choleraesuis as a gene delivery vector. In this study, we exploited S. choleraesuis carrying thrombospondin-1 (TSP-1) gene for treating primary melanoma and experimental pulmonary metastasis in the syngeneic murine B16F10 melanoma model. Systemic administration of S. choleraesuis allowed targeted gene delivery to tumors. The bacteria accumulated preferentially in tumors over livers and spleens at ratios ranging from 1000:1 to 10,000:1. The level of transgene expression via S. choleraesuis-mediated gene transfer in tumors could reach more than 1800-fold higher than in livers and spleens. Notably, bacterial accumulation was also observed in the lungs with metastatic nodules, but not in healthy lungs. When administered into mice bearing subcutaneous or pulmonary metastatic melanomas, S. choleraesuis carrying TSP-1 gene significantly inhibited tumor growth and enhanced survival of the mice. Immunohistochemical studies in the tumors from these mice displayed decreased intratumoral microvessel density. Taken together, these findings suggest that TSP-1 gene therapy delivered by S. choleraesuis may be effective for the treatment of primary as well as metastatic melanomas.     

Antitumor efficacy of a novel polymer-peptide-drug conjugate in human tumor xenograft models

We have designed a new dextran-peptide-methotrexate conjugate to achieve tumor-targeted delivery of chemotherapeutics. The dextran carrier was selected to allow passive targeting and enhanced permeation and retention (EPR). The peptide linker has also been optimized to allow drug release in the presence of matrix-metalloproteinase-2 (MMP-2) and matrix-metalloproteinase-9 (MMP-9), 2 important tumor-associated enzymes. The new conjugate was assessed for its in vivo antitumor efficacy and systemic side effects. It was compared with free methotrexate (MTX) and a similar conjugate, differing by an MMP-insensitive linker, at equivalent intraperitoneal dosages. The MMP-sensitive conjugate demonstrated tolerable in vivo side effects and effective inhibition of in vivo tumor growth by 83% in each of the 2 separate tumor models that overexpress MMP (HT-1080 and U-87). The antiproliferative effect of the drug contributed to the inhibition of tumor growth. In contrast, free MTX resulted in no significant tumor reduction in the same models. Neither free MTX nor the conjugate caused any tumor inhibition in the mice bearing RT-112, a slower growing model that does not overexpress MMP. MMP-insensitive conjugates, though able to inhibit tumor growth, caused toxicity in the small intestine and bone marrow.     

Antitumor effect of Cepharanthin in the double grafted tumor system

The antitumor effect of Cepharanthin (CR) in a new experimental mouse model was studied. Intratumoral administration of CR strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumor. Subsequently, the antimetastatic effect of CR was examined in the double grafted tumor system, in which mice first received simultaneous intradermal inoculations of Meth-A in both right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with 0.5 mg of CR in the right tumor on days 3, 4 and 5. CR inhibited the growth of not only the right but also the left, nontreated tumor. Immunized spleen cells were taken from mice which had been cured with the intratumoral administration of CR. Adoptive transfer of CR immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 antibody. These results suggest that intratumoral administration of CR might induce Lyt-1 positive cytotoxic cells in the spleen and the left, non-treated tumor. In BALB/c nude mice, CR inhibited the growth of the right tumor but did not the left tumor. Therefore, the antitumor activity of CR on the left tumor in the double grafted tumor system is associated with a sequential immune mechanism in which T cells may play an important role. The antitumor effect of CR on the right tumor is direct cytotoxic effect. TILs (tumor infiltrating lymphocytes) obtained from left and right sides tumors treated with CR were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TILs from both sides clearly inhibited the growth of admixed Meth-A cells and seems to play an important role on antitumor effect in both tumors.     

Antitumor effect of carboplatin on in vitro three-dimensional tumor]

We studied the antitumor effect of Carboplatin on the three-dimensional tumor in vitro. First, we observed the three-dimensional growth of cultured tumor cell lines (Hep 2, KB and HT 29 cells), which were developed in the combined culture system of fibrin matrix and agar culture. The tumor cells developed a 3 x 3 mm tumor in diameter in the in vitro 10 day-culture system. This size was large enough to allow the histologic study. When we applied Carboplatin to the three-dimensional tumors, histologic change of the three-dimensional tumor developed from Hep 2 and KB cells were observed in a dose dependent manner. However, no remarkable histologic change was observed in the three-dimensional tumor developed from HT 29 cells. These results suggest the experimental system of three-dimensional tumor can predict histologically the antitumor effect of Carboplatin on various tumors at the solid tumor level.     

Antitumor activity of erlotinib in combination with capecitabine in human tumor xenograft models

Purpose: To examine the antitumor activity and tolerability of a combination comprising erlotinib and capecitabine in human colorectal, breast and epidermal cancer xenograft models. Further aims of the study were to examine the effects of single-agent erlotinib therapy on tumor growth, and on thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) levels, (enzymes which activate and deactivate capecitabine, respectively) in tumor tissue. Methods: BALB/c nu/nu mice bearing LoVo and HT-29 (colon cancer), A-431 (vulval cancer), and KPL-4 and MAXF401 (breast cancer) human tumors were treated with erlotinib 100 mg/kg/day and/or capecitabine 359 or 90 mg/kg/day, by oral administration once daily for 14 days. Results: The maximum tolerated dose (MTD) of erlotinib, formulated in carboxymethylcelluose/Tween 80, was identified as 125 mg/kg/day. Erlotinib at a dose of 100 mg/kg/day achieved significant tumor-growth inhibition in the, LoVo, KPL-4, and A-431 models. Some inhibition of MAXF401 tumor growth was observed, but was not significant. In the HT-29 model, erlotinib showed less marked but statistically significant antitumor activity. On day 15, mean tumor-growth inhibition in HT-29, LoVo, KPL-4, MAXF401, and A-431 models was 46, 74, 71, 20, and 85%, respectively. Evaluation of erlotinib/capecitabine combination therapy, at sub-optimal doses, in the three erlotinib-sensitive tumor models LoVo, KPL-4 and A-431, demonstrated at least additive activity with the combination compared with the single agents. In the A-431 and LoVo models, the combination of agents had greater antitumor activity than the single agent capecitabine alone at the MTD. Erlotinib in combination with capecitabine was not associated with significantly increased toxicity compared with single-agent therapy. Erlotinib 100 mg/kg/day induced significant upregulation of TP and DPD in the LoVo model, a significant upregulation of TP in the HT-29, MAXF401 and A-431 models, but had no obvious effect on TP and DPD levels in the KPL-4 model. In the A-431 model, selective upregulation of TP by erlotinib 100 mg/kg resulted in an increased TP:DPD ratio. In the LoVo model, immunohistochemistry revealed marked upregulation of TP (but not DPD by erlotinib). Conclusions: Erlotinib inhibits tumor growth in a range of human tumor xenograft models, including breast and colorectal cancer (CRC). Erlotinib and capecitabine demonstrated at least additive activity in LoVo, KPL-4 and A-431 tumor models. The antitumor activity of the combination was greater than that of capecitabine alone at the MTD. Erlotinib treatment did affect the TP in the CRC tumor models as confirmed immunohistochemically. The findings of this study support clinical evaluation of erlotinib, both as a single agent and in combination with capecitabine, for the treatment of CRC and breast cancer.