Strobilanthes crispus bioactive subfraction inhibits tumor progression and improves hematological and morphological parameters in mouse mammary carcinoma model.

ETHNOPHARMACOLOGICAL RELEVANCE: Locally known as 'pecah batu', 'bayam karang', 'keci beling' or 'batu jin', the Malaysian medicinal herb, Strobilanthes crispus (S. crispus), is traditionally used by the local communities as alternative or adjuvant remedy for cancer and other ailments and to boost the immune system. S. crispus has demonstrated multiple anticancer therapeutic potential in vitro and in vivo. A pharmacologically active fraction of S. crispus has been identified and termed as F3. Major constituents profiled in F3 include lutein and ß-sitosterol. AIM OF THE STUDY: In this study, the effects of F3, lutein and ß-sitosterol on tumor development and metastasis were investigated in 4T1-induced mouse mammary carcinoma model. MATERIALS AND METHODS: Tumor-bearing mice were fed with F3 (100 mg/kg/day), lutein (50 mg/kg/day) and ß-sitosterol (50 mg/kg/day) for 30 days (n = 5 each group). Tumor physical growth parameters, animal body weight and development of secondary tumors were investigated. The safety profile of F3 was assessed using hematological and histomorphological changes on the major organs in normal control mice (NM). RESULTS: Our findings revealed significant reduction of physical tumor growth parameters in all tumor-bearing mice treated with F3 (TM-F3), lutein (TM-L) or ß-sitosterol (TM-ß) as compared with the untreated group (TM). Statistically significant reduction in body weight was observed in TM compared to the NM or treated (TM-F3, TM-L and TM-ß) groups. Histomorphological examination of tissue sections from the F3-treated group showed normal features of the vital organs (i.e., liver, kidneys, lungs and spleen) which were similar to those of NM. Administration of F3 to NM mice (NM-F3) did not cause significant changes in full blood count values. CONCLUSION: F3 significantly reduced the total tumor burden and prevented secondary tumor development in metastatic breast cancer without significant toxicities in 4T1-induced mouse mammary carcinoma model. The current study provides further support for therapeutic development of F3 with further pharmacokinetics studies.

Inhibition of breast tumor growth in mice after treatment with ceramide analog 315.

The aim of this study was to evaluate the anticancer and antitumor activities of ceramide analog 315 in nude mice. Nude mice (n=10) were injected bilaterally with 5×10 MDA-MB-231 cells on each side. Tumors were allowed to form for 2 weeks. The mice were then divided into two groups (n=5 in each group). The control group mice were injected with 25 µl of dimethyl sulfoxide and the treatment group mice were injected with 10 mg/kg of analog 315 (in dimethyl sulfoxide, 25 µl volume) every day for a period of 3 weeks. Animal weights and tumors were measured every week for 3 weeks. At the end of the experimental period, control animals had retained excess fluid, and showed larger tumor sizes compared with the treated group (2.95 vs. 1.67 g). A 45% reduction in tumor size and 80% decrease in tumor volume were observed in the treatment group. There was a significant increase in the weights of liver (10%) and spleen (19%) between the control and treated animals. Hematoxylin and Eosin staining of MDA-MB-231 tumor sections revealed more acellular necrotic regions in tumors from the treatment groups compared with the ones from the control group. Ki67, a proliferation marker was higher in number in control tumor section (71.8±12.8) compared to the treatment tumor section (37.4±10.4) (P<0.001). Photomicrographs showed metastatic tumor burden in kidney, lungs, and spleen collected from the control group mice bearing MDA-MB-231 tumors. Treatment group mice showed normal microscopic tissue architecture. Overall, our study showed tumor growth inhibition and antimetastatic effects for the novel ceramide analog 315.

Protective mechanisms of purified polyphenols from pinecones of Pinus koraiensis on spleen tissues in tumor-bearing S180 mice in vivo.

The previous study evaluated the antitumor activity and the underlying mechanism of the purified polyphenols from pinecones of Pinus koraiensis (PPP-40) using a tumor-bearing S180 mice model. This study was designed to evaluate the protective effects of PPP-40 on spleen tissues of S180 mice in vivo. Pretreatment with PPP-40 (150 mg per kg BW per D) could significantly inhibit tumor growth, enhance spleen index and prevent the decline of haematological parameters of S180 mice induced by the tumor microenvironment. Moreover, the treatment with PPP-40 was shown to significantly inhibit splenocyte apoptosis by TUNEL staining and flow cytometry, characterized by the inhibition of splenocyte cycle (G0/G1) arrest, increase in the percentages of splenic T lymphocytes (CD3+ T cells) and T cell subsets (CD3+CD4+ and CD3+CD8+ T cells), as well as the production of T cell-related cytokines (IL-2, IL-12, and TNF-α) in splenocytes exposed to the tumor microenvironment. These effects were associated with a decrease in oxidative stress, as evidenced by the changes in the SOD, GSH-Px, GSH and MDA levels of liver and spleen tissues of S180 mice. Furthermore, the protective effect of PPP-40 on spleen tissues was deeply analyzed by detecting apoptosis-related proteins using immunohistochemistry staining. The results indicated that the protective multi-mechanisms of action also were associated with the inhibition of apoptosis through down-regulation protein expressions of Bax, caspase-9, caspase-8 caspase-3, Fas and up-regulation of the expressions of Bcl-2. These results suggested that PPP-40 is a natural antitumor agent and possesses strong immunomodulatory activities by protecting the spleen tissues of tumor-bearing S180 mice.

Oral inoculation of probiotics Lactobacillus acidophilus NCFM suppresses tumour growth both in segmental orthotopic colon cancer and extra-intestinal tissue.

Modulation of the cellular response by the administration of probiotic bacteria may be an effective strategy for preventing or inhibiting tumour growth. We orally pre-inoculated mice with probiotics Lactobacillus acidophilus NCFM (La) for 14 d. Subcutaneous dorsal-flank tumours and segmental orthotopic colon cancers were implanted into mice using CT-26 murine colon adenocarcinoma cells. On day 28 after tumour initiation, the lamina propria of the colon, mesenteric lymph nodes (MLN) and spleen were harvested and purified for flow cytometry and mRNA analyses. We demonstrated that La pre-inoculation reduced tumour volume growth by 50·3 %, compared with untreated mice at 28 d after tumour implants (2465·5 (SEM 1290·4) v. 4950·9 (SEM 1689·3) mm³, P<0·001). Inoculation with La reduced the severity of colonic carcinogenesis caused by CT-26 cells, such as level of colonic involvement and structural abnormality of epithelial/crypt damage. Moreover, La enhanced apoptosis of CT-26 cells both in dorsal-flank tumour and segmental orthotopic colon cancer, and the mean counts of apoptotic body were higher in mice pre-inoculated with La (P<0·05) compared with untreated mice. La pre-inoculation down-regulated the CXCR4 mRNA expressions in the colon, MLN and extra-intestinal tissue, compared with untreated mice (P<0·05). In addition, La pre-inoculation reduced the mean fluorescence index of MHC class I (H-2Dd, -Kd and -Ld) in flow cytometry analysis. Taken together, these findings suggest that probiotics La may play a role in attenuating tumour growth during CT-26 cell carcinogenesis. The down-regulated expression of CXCR4 mRNA and MHC class I, as well as increasing apoptosis in tumour tissue, indicated that La may be associated with modulating the cellular response triggered by colon carcinogenesis.

Medial pancreatectomy for a neuroendocrine tumor invading the splenic artery and vein.

CONTEXT: Pancreatic tumors in the midportion have traditionally been treated by an extended right or left pancreatectomy. A medial or central pancreatectomy is an alternative technique for benign or low-grade malignant neoplasms located to the left of the gastroduodenal artery and close to the splenomesenteric confluence. CASE REPORT: A 38-year-old woman with no previous surgical history presented with epigastric abdominal pain. A computed tomography scan showed a 4 cm heterogeneous lesion within the pancreatic body. This tumor invaded the splenic artery and vein. There was no postoperative diabetes mellitus or exocrine insufficiency. The patient continues to be well after a 10-month follow-up without pancreatic insufficiency or local recurrence, and CT has demonstrated splenic perfusion by the collateral vessels. CONCLUSION: We believe that a medial or central pancreatectomy may be a safe procedure where there is involvement of the large splenic vessels by a low grade malignant pancreatic tumor and that a systematic splenectomy is not justified.

Suppression of tumorigenesis and metastasis of hepatocellular carcinoma by shRNA interference targeting on homeoprotein Six1.

We previously demonstrated that the overexpression of homeoprotein Six1 in hepatocellular carcinoma (HCC) patients is associated with venous infiltration, advanced pathologic tumor metastasis (pTNM) stage and poor overall survival rate (Ng et al. Br J Cancer 2006;95:1050-5). In this study, short hairpin RNA (shRNA) interference approach was used to suppress the expression of Six1 in a metastatic HCC cell line MHCC97L. Stable transfectant MHCC97L-shSix1 carrying Six1-specific shRNA plasmid was established to downregulate Six1 expression to about 40% when compared with MHCC97L-Control. In vitro functional assays demonstrated that the growth rate and proliferation ability of MHCC97L-shSix1 cells were markedly decreased. Moreover, significant decrease of cell motility and invasiveness were observed in MHCC97L-shSix1 cells. Data from in vivo xenograft tumorigenesis model demonstrated that the size of tumor in MHCC97L-shSix1 group was dramatically reduced. Experimental and spontaneous metastasis models indicated that targeting Six1 suppression noticeably reduced the pulmonary metastasis in MHCC97L-shSix1 group. To identify Six1-regulated targets, cDNA microarray was employed to compare the expression profiles of MHCC97L-Control and MHCC97L-shSix1 cells. Twenty-eight downregulated and 24 upregulated genes with known functions were identified in MHCC97L-shSix1. The functions of these target genes are involved in diverse biological activities. Our data suggest that Six1 may be involved in regulation of proliferation and invasiveness of HCC; thus targeting suppression of Six1 is a viable option for treating HCC patients.

Liposomal gemcitabine (GemLip)-efficient drug against hormone-refractory Du145 and PC-3 prostate cancer xenografts.

BACKGROUND: Gemcitabine (Gemc) is an efficient chemotherapeutic drug in various cancer types (e.g., pancreas) but has only limited effects on hormone-refractory prostate cancer (HRPCa). Since HRPCa cells are highly sensitive to even low doses of Gemc in vitro, the lack of clinical effects might be due to rapid degradation of Gemc by deaminases combined with impaired accumulation in tumor tissue and PCa cells. Liposomal formulation (GemLip) is expected to protect the entrapped cytotoxic substance from enzymatic degradation and furthermore augment its accumulation within tumor tissues due to an enhanced permeability of the tumor vessels. METHODS: Anti-tumoral and anti-metastatic activity of GemLip and Gemc were investigated in two luciferase-expressing, human hormone-refractory PC-3 and Du145 HRPCa xenograft models in immunodeficient mice. Tumor growth was monitored by in vivo luminescence imaging (orthotopic) or callipering (subcutaneous). Anti-metastatic effects of treatment were determined by in vitro luciferase assay of the tissues. RESULTS: Tumor growth of subcutaneous Du145 xenografts was significantly inhibited only by GemLip (8 mg/kg: P = 0.014 and 6 mg/kg: P = 0.011) but not by conventional Gemc (360 mg/kg). In contrast, growth of orthotopic PC-3 xenografts was significantly inhibited by both, GemLip (P = 0.041) and Gemc (P = 0.002). The drugs furthermore strongly reduced spleen and liver metastases in this model. CONCLUSIONS: As shown by the very low efficient concentration of GemLip, liposomal entrapment of Gemc greatly enhances its activity. GemLip has, even at very low doses, a significant anti-tumoral and anti-metastatic therapeutic effect in HRPCa xenografts in vivo and was beneficial even when the conventional Gemc failed.

The metastatic T-cell hybridoma antigen/P-selectin glycoprotein ligand 1 is required for hematogenous metastasis of lymphomas.

Using variants of the murine BW5147 lymphoma cell-line, we have previously identified 3 monoclonal antibodies (MAbs) that discriminate between metastatic and nonmetastatic BW5147-derived T-cell hybridomas and lymphomas, as well as BW5147-unrelated T-lymphomas. These MAbs were reported to recognize an identical membrane-associated sialoglycoprotein, termed "metastatic T-cell hybridoma antigen" (MTH-Ag). Here, we document that the expression pattern of the MTH-Ag on metastatic and nonmetastatic BW5147 variants correlates with that of the P-selectin glycoprotein ligand 1 (PSGL-1), a sialomucin involved in leukocyte recruitment to sites of inflammation. Moreover, the MAbs against the MTH-Ag recognize PSGL-1 when it is transfected in MTH-Ag-negative BW5147 variants, suggesting that the MTH-Ag is PSGL-1. Overexpression of MTH-Ag/PSGL-1 in MTH-Ag-negative BW5147 variants did not affect their in vivo malignancy. Yet, down-regulation of MTH-Ag/PSGL-1 expression on metastatic, MTH-Ag-positive BW5147 variants, using an RNA interference (RNAi) approach, resulted, in a dose-dependent manner, in a significant reduction of liver and spleen colonization and a delay in mortality of the recipient mice upon intravenous inoculation. Collectively, these results demonstrate that, although MTH-Ag/PSGL-1 overexpression alone may not be sufficient for successful dissemination and organ colonization, MTH-Ag/PSGL-1 plays a critical role in hematogenous metastasis of lymphoid cancer cells.

Embryonic stem cells develop into hepatocytes after intrasplenic transplantation in CCl4-treated mice.

AIM: To transplant undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl4)-treated mice to determine their ability to differentiate into hepatocytes in the liver. METHODS: CCl4, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 (n=12), 1 x 10(5) undifferentiated ES cells (0.1 mL of 1 x 10(6)/mL solution), genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice (n=12) were injected with 0.2 mL of saline twice a week, instead of CCl4, and the same amount of ES cells was transplanted into the spleens. Group 3 mice (n=6) were treated with CCl4 and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d (PD) 10, 20, and 30. RESULTS: Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice (CCl4-treated) on PD 10, however, not in those untreated with CCl4 (group 2). The GFP-positive cells were also immunopositive for albumin (ALB), alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantation during the experimental period. CONCLUSION: Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.

IL-12 cDNA direct injection: antimetastatic effect from a single injection in a murine hepatic metastases model.

BACKGROUND: Interleukin 12 (IL-12) gene therapy is an effective antitumor agent in local and metastatic murine tumor models. We sought to evaluate the antimetastatic effect of IL-12 cDNA in a liver metastases model. MATERIALS AND METHODS: A liver metastases model was induced by creating a "primary" splenic tumor through inoculation of 1 x 10(5) TS/A adenocarcinoma cells directly into the inferior pole of the spleen in female BALB/c mice. On day 4, 50 microg of IL-12 cDNA or control plasmid DNA was injected into splenic tumor, followed by splenectomy on day 8. Mice were sacrificed on day 25 to assess liver tumor burden. IL-12 mRNA and mIL-12 and IFN-gamma protein levels were assessed after IL-12 injection. Peripheral blood CD4+, CD8+, and NK cells were quantified on day 14 using FACS. To determine the significance of site of cytokine DNA injection, IL-12 cDNA was injected on day 4 into splenic tumor or into the non-involved spleen after isolation of the inferior and superior portions of the spleen, respectively, with surgical clips. Splenectomy was performed on day 8 and sacrifice was performed on day 25. RESULTS: IL-12 mRNA was detected in the liver 8 h after injection, with a peak at 24 h. After splenic injection, protein levels of IL-12 and IFN-gamma were detectable in the liver and spleen 24 h after treatment. IL-12 and IFN-gamma were not detectable in control animals. In the peripheral blood, there was a marked increase in NK cells (13% of total lymphocytes versus 4%, control) and in the CD4+/CD8+ ratio (5.5 versus 1.9). At day 25, there was a marked antimetastatic effect after IL-12 injection into either splenic tumor [liver:body weight, 6.2 versus 10.9 (control), P = 0.007] or non-involved spleen (6.8 g versus 10.7 g, P = 0.005). There was no difference in the antimetastatic effect between animals injected into splenic tumor or non-involved spleen (P = 0.3). CONCLUSION: Injection with a single dose of IL-12 cDNA into splenic tumor or non-involved spleen resulted in a profound antimetastatic effect. Splenic IL-12 injection results in mRNA expression in the liver, protein expression in the liver and spleen, and a marked increase in NK cells and the CD4+/CD8+ ratio in peripheral blood.

The immunization site of cytokine-secreting tumor cell vaccines influences the trafficking of tumor-specific T lymphocytes and antitumor efficacy against regional tumors.

Tumor cells engineered to secrete cytokines, referred to as tumor cell vaccines, can often generate systemic antitumor immunity and, in many cases, cause tumor regression. We compared the efficacy of s.c. immunization or intrahepatic immunization of GM-CSF-expressing tumor cell vaccines on the growth of s.c. or orthotopic liver tumors. A chemically transformed hepatic epithelial cell line, GP7TB, derived from Fischer 344 rats, was used to generate tumor models and tumor cell vaccines. Our results demonstrated that two s.c. injections of an irradiated tumor cell vaccine significantly controlled the growth of s.c. tumors, but was completely ineffective against orthotopic liver tumors. Effector cell infiltration in liver tumors was markedly reduced compared with s.c. tumors. Enhanced apoptosis of some effector cells was observed in the liver tumors compared with the s.c. tumors. Furthermore, the T cells induced by s.c. immunization preferentially migrated to s.c. tumor sites, as demonstrated by adoptive transfer experiments. In contrast, intrahepatic immunization, using parental tumor cells admixed with adenoviruses carrying the GM-CSF gene, yielded significantly better therapeutic effects on the liver tumors than on the s.c. tumors. Adoptive transfer experiments further confirmed that the T cells induced by liver immunization preferentially migrated to the liver tumor sites. Our results demonstrate that distinct T cell populations are induced by different immunization routes. Thus, the homing behavior of T cells depends on the route of immunization and is an important factor determining the efficacy of immunotherapy for regional tumors.

Inhibitory effect of magnolol on tumour metastasis in mice.

It has previously been reported that magnolol, a phenolic compound isolated from Magnolia obovata, inhibited tumour cell invasion in vitro. The purpose of this study was to investigate the antimetastatic effect of magnolol on tumour metastasis in vivo with experimental and spontaneous metastasis models and to clarify the mechanism. The antimetastatic effects of magnolol were evaluated by an experimental liver and spleen metastasis model using L5178Y-ML25 lymphoma, or an experimental and spontaneous lung metastasis model using B16-BL6 melanoma. Intraperitoneal (i.p.) administration of 2 or 10 mg/kg of magnolol significantly suppressed liver and spleen metastasis or lung metastasis. As for the spontaneous lung metastasis model using B16-BL6 melanoma, multiple i.p. administrations of 10 mg/kg of magnolol after and before tumour inoculation significantly suppressed lung metastasis and primary tumour growth. In addition, magnolol significantly inhibited B16-BL6 cell invasion of the reconstituted basement membrane (Matrigel, MG) without affecting cell growth. These data from the in vivo experiments suggest that magnolol possesses strong antimetastatic ability and that it may be a lead compound for drug development. The antimetastatic action of magnolol is considered to be due to its ability to inhibit tumour cell invasion.

Src family kinase inhibitor PP2 restores the E-cadherin/catenin cell adhesion system in human cancer cells and reduces cancer metastasis.

The E-cadherin/catenin cell adhesion system is often down-regulatedin epithelial tumors. This is thought to play an important role in cancer invasion and metastasis. Restoring this system may enable suppression of the metastatic spread of cancer. This study examined the effect of Src family kinase inhibitor PP2 on E-cadherin-mediated cell-cell adhesion and metastatic potentials. In cell aggregation assays, PP2 stimulated the aggregation of colon, liver, and breast cancer cells. In vitro cultures of cancer cells showed that PP2 induced strong cell-cell contact. Immunoblot analysis showed that PP2 enhanced E-cadherin/catenin expression and that increased E-cadherin/catenin proteins were strongly associated with the actin cytoskeleton. Northern blot studies indicated that the observed increase of E-cadherin/catenin protein content was due to their increased gene expression. After the spleens of severe combined immunodeficient mice were inoculated with cancer cells, treatment with PP2 for 3 weeks markedly reduced the rate of liver metastasis, compared with the control counterparts. Our data demonstrate that PP2 can activate the functioning of the E-cadherin-mediated cell adhesion system, which is associated with the suppression of metastasis in cancer cells. Thus, selective inhibition of Src activation may be potentially useful in the prevention of cancer metastasis.

A new type of antimetastatic peptide derived from fibronectin.

PURPOSE: We found previously that fibronectin (FN) has a cryptic functional site (YTIYVIAL sequence within the 14th type III repeat) opposing cell adhesion to extracellular matrix. A 22-mer FN peptide containing this site, termed FNIII14, inhibits beta1 integrin-mediated adhesion without binding to integrins. The present study shows that FNIII14 has the potential to prevent lymphoma cell metastasis. EXPERIMENTAL DESIGN: Antimetastatic effect of FNIII14 has been evaluated through in vitro or in vivo experiments. RESULTS: FNIII14 inhibited the integrin alpha4beta1-mediated B lymphoma Ramos cell adhesion to VCAM-1 on venule endothelial cells, as well as to FN. Murine T lymphoma L5178Y-ML25 cells, which are known to metastasize to liver and spleen, preferentially adhered to vitronectin (VN) and migrated toward VN concentration gradients. FNIII14 abrogated both the integrin alphavbeta3-mediated adhesion and migration of L5178Y-ML25 cells. Inhibition of the alphavbeta3mediated L5178Y-ML25 cell adhesion by FNIII14 was reversed by phenylarsine oxide, a protein tyrosine phosphatase inhibitor. In addition, FNIII14 abrogated the VN-stimulated tyrosine phosphorylation of intracellular signaling proteins, including focal adhesion kinase (p125(FAK)) and paxillin, suggesting that such a diversity of FNIII14 effects might be because of the negative regulation of p125(FAK) and paxillin tyrosine phosphorylation, which has been involved in adhesion signals transduced by different integrins. The in vivo experiment using a murine metastasis model showed that FNIII14 would inhibit liver and spleen metastases of L5178Y-ML25 cells at a dose much lower than that of RGDS. CONCLUSIONS: FNIII14 might be applicable as a new type of antimetastatic agent distinct from integrin-binding peptides.

Prophylactic effect of Korean mistletoe (Viscum album coloratum) extract on tumor metastasis is mediated by enhancement of NK cell activity.

We here demonstrated the prophylactic effect of an extract (KM-110) from Viscum album coloratum, a Korean mistletoe, on tumor metastasis produced by highly metastatic tumor cells, colon 26-M3.1 carcinoma, B16-BL6 melanoma and L5178Y-ML25 lymphoma cells, using experimental models in mice. Intravenous (i.v.) administration of KM-110 (100 microg/mouse) 2 days before tumor inoculation significantly inhibited lung metastasis of B16-BL6 and colon 26-M3.1 cells, and liver and spleen metastasis of L5178Y-ML25 cells. The prophylactic effect of KM-110 on tumor metastasis was evident with various administration routes, i.e. subcutaneous, oral, intranasal as well as i.v., and was dependent upon the dose of KM-110 administered. Furthermore, mice given KM-110 (100 microg) 2 days before tumor inoculation showed significantly prolonged survival rates compared with the untreated mice. In a time course analysis of NK activity, i.v. administration of KM-110 (100 microg) significantly augmented NK cytotoxicity to Yac-a tumor cells from 1 to 3 days after KM-110 treatment. Furthermore, depletion NK cells by injection of rabbit anti-asialo GM1 serum completely abolished the inhibitory effect of KM-110 on lung metastasis of colon 26-M3.1 cells. These results suggest that KM-110 possesses immunopotentiating activity which enhances the host defense system against tumors, and that its prophylactic effect on tumor metastasis is mediated by NK cell activation.

TAC-101, a benzoic acid derivative, inhibits liver metastasis of human gastrointestinal cancer and prolongs the life-span.

We examined the anti-tumor effect of a novel benzoic acid derivative, TAC-101 (4-[3,5-bis(trimethylsilyl) benzamide] benzoic acid) on models with liver metastasis. Oral administration of TAC-101 significantly inhibited spontaneous liver metastasis of AZ-521 (human gastric cancer ) by orthotopic implantation to athymic nude mice. It also inhibited both the liver metastasis of AZ-521 induced by intrasplenic injection and the secondary lung metastasis from the liver. In addition, TAC-101 inhibited the proliferation of Co-3 (human colon adenocarcinoma) that formed a single nodule in the liver of athymic nude mice by intrahepatic implantation. The growth inhibitory effect of TAC-101 on AZ-521 experimental liver metastasis was observed when treatment was started on day 7, 14, or 21 which may correspond to the progressive stage of liver metastasis in clinical settings. Multiple administration of TAC-101 (8 mg/kg/day) significantly prolonged survival time of the animals with liver metastasis by intrasplenic injection of AZ-521 (T/C = 230%) and A549 (human lung adenocarcinoma; T/C = 186%). These effects of TAC-101 were stronger than those of 5-FU, CDDP or ATRA. Furthermore, TAC-101 inhibited the binding of AP-1 to DNA on electrophoretic mobility shift assay using nuclear extract of AZ-521 cells, although ATRA did not inhibit. These findings suggested that TAC-101 may be a candidate for a new class of anti-cancer agents for liver metastasis.

GD1alpha-replica peptides functionally mimic GD1alpha, an adhesion molecule of metastatic tumor cells, and suppress the tumor metastasis.

A novel peptide technology to produce mimicking peptides of carbohydrate moiety (which we propose to name glyco-replica peptides) is a useful tool to elucidate the functions of glycoconjugate. Carbohydrate moiety of ganglioside GD1alpha functions as a molecule involved in the adhesion between murine highly metastatic lymphoma RAW117-H10 cells and hepatic sinusoidal endothelial (HSE) cells. To prepare peptides which mimic the carbohydrate structure of GD1alpha, phage clones expressing peptides which bound to a monoclonal antibody against GD1alpha (KA17) were isolated from a phage-displayed random peptide library. Four phage clones having affinity to the monoclonal antibody KA17 were isolated, and these clones showed inhibitory effect on the binding of KA17 to GD1alpha. The amino acid sequences of the displayed pentadecamers were determined, and one of the phages displaying sequence WHWRHRIPLQLAAGR bound to HSE cells directly and showed the highest inhibitory effect on the adhesion between RAW117-H10 cells and HSE cells. The synthesized peptides having the same sequences to the displayed 15mers in the four isolated phage clones also showed the inhibitory effect on the adhesion of RAW117-H10 cells to HSE cells, and, again, the WHWRHRIPLQLAAGR peptide showed the highest inhibitory effect. Furthermore, intravenous injection of the peptide brought almost complete inhibition of the metastasis of RAW117-H10 cells to lung and spleen, and about 50% inhibition of the liver metastasis. These results indicate that GD1alpha plays an important role for metastasis of RAW117-H10 cells, and the peptides obtained by the present procedure are able to mimic the functional role of the glycoconjugate.

Antitumor effect of DX-8951, a novel camptothecin analog, on human pancreatic tumor cells and their CPT-11-resistant variants cultured in vitro and xenografted into nude mice.

DX-8951 is a novel water-soluble derivative of camptothecin. We evaluated the effects of DX-8951 on the growth of several pancreatic tumor cell lines in vitro and in vivo. In vitro cytotoxic activity of DX-8951 against SUIT-2 and KP-1N cells, as indicated by IC50 value, was several times more potent than that of SN-38, an active metabolite of CPT-11, and dozens of times more potent than that of SK&F104864 (topotecan). DX-8951 also showed the greatest cytotoxicity against CPT-11-resistant variants, SUIT-2/CPT-11 and KP-1N/CPT-11 cells, and the cross-resistance of these cells to DX-8951 was lower than that to SN-38 and SK&F104864. Topoisomerase I inhibitory activity of DX-8951 was about three-fold stronger than that of SN-38, as measured in crude nuclear extract obtained from SUIT-2 cells. DX-8951 induced DNA fragmentation, a specific feature of apoptosis, in SUIT-2 cells more effectively than SN-38. DX-8951 exhibited potent antitumor effects against SUIT-2 in a solid tumor model and in a liver metastasis model, in which tumor cells were xenografted subcutaneously and intrasplenically, respectively, into nude mice. The in vivo effects were closely similar to or somewhat superior to those of CPT-11. DX-8951 also showed significant antitumor effects against SUIT-2/CPT-11 solid tumors, against which CPT-11 had no effect. These results suggest that, on the basis of its strong antitumor activity and effectiveness against CPT-11-resistant tumors, DX-8951 may be a useful therapeutic agent in the treatment of human cancer. The potent cytotoxicity of DX-8951 may result from strong inhibition of topoisomerase I, which may then trigger apoptotic cell death.

Experimental radiotherapy and metastasis of human lung cancer xenografts in nude mice.

Using a cell line derived from human lung cancer (AOI), we successfully established human xenografts in KSN nude mice, which showed high incidence of multiple spontaneous metastases. The highest incidence of metastasis in untreated hosts was observed in the spleen followed by the lungs and lymph nodes. The rate of metastasis reached 100% in the mice bearing large sized tumors, when metastasis to any organ or tissue was counted. Experimental radiotherapy caused remarkable redistribution of metastatic foci among different organs. Lung metastasis tended to decrease, while metastasis to the liver and the kidney was increased after radiotherapy. Radiation-induced production of cytokines was speculated to be responsible for such an alteration of metastasis pattern.