Anti-angiogenic activity of arachidonic acid metabolism inhibitors in angiogenesis model systems involving human microvascular endothelial cells and neovascularization in mice

We have established an in vitro angiogenesis model using human omental microvascular endothelial (HOME) cells, in which epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-α) stimulated cell migration and tube formation. In this study, we examined whether α-guaiaconic acid (GR-12) and its synthetic 20 derivatives showed inhibition of cell migration and tubular formation of HOME cells. We found that GR-12 inhibits arachidonic acid metabolism, while GR-12 and one derivative, GS-01, inhibit tubular formation of endothelial cells in our model system. Confluent monolayers of HOME cells were damaged with a razor blade and incubated with or without TGF-α; HOME cell migration was stimulated about 1.5-fold over control values in the presence of TGF-α. Treatment of HOME cells with GR-12 or GS-01 inhibited both spontaneous and TGF-α-stimulated migration. GR-12 or GS-01 inhibited TGF-α-induced HOME-cell tube formation in type-1 collagen gels. We examined whether these compounds could modulate tubular formation of HOME cells induced by human cancer cells. Enhanced tube formation of HOME cells by co-cultured esophageal cancer cells was almost completely inhibited by co-administration of GR-12 or GS-01. Both compounds also inhibited formation of tubular networks of HOME cells on Matrigels. We also examined anti-angiogenic activity of these compounds in an in vivo model system of tumor angiogenesis in mice. In this system, GS-01 inhibited development of capillary networks at a rate comparable to that of a well-known anti-angiogenic compound, fumagillin, but GR-12 did not. The inhibitor of arachidonic acid metabolism is thus expected to modulate tumor angiogenesis.     

口腔鳞癌术前应用平阳霉素的近期疗效观察

本文作者对术前应用平阳霉素治疗的５２例口腔鳞癌患者进行观察，通过对肿瘤体积大小变化的测量，发现化疗的疗效和肿瘤的分化程度、生长方式，有无副作用以及某些未知因素有关，另外作者还发现平阳霉素用量达１２０ｍｇ左右时，其大小变化最大，以后瘤体的大小变化就不显著，作者认为１２０ｍｇ的剂量可以作为术前化疗剂量的一个标准。     

Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice

The effects of estradiol and tamoxifen (TAM) on the estrogen-dependent human breast cancer cell line MCF-7 grown in vitro and in nude mice were compared. The effect on growth was determined by cell number in vitro and by tumor growth curves in nude mice. The effects on the cell cycle kinetics were determined by repeated flow cytometric DNA analyses in vitro and in vivo and by the technique of labeled mitosis in nude mouse-grown tumors. Under in vitro conditions, estradiol induced a pronounced increase in S-phase fraction and cell number. TAM inhibited growth of MCF-7 cells with a concomitant increase in the G1 phase from 60% to 75%. In nude mice, MCF-7 only formed tumors in estradiol-supplemented mice. No differences were observed in growth and cell kinetics between 0.1 and 1.0 mg of estradiol. Daily i.p. injections of TAM resulted in tumor growth inhibition with shrinkage of tumors. The flow cytometric DNA analysis and percentage of labeled mitosis investigations revealed no significant differences in the proliferation kinetics of TAM-treated and control tumors. Calculating the cell loss factor demonstrated an increase from 69% in control tumors to 107% in TAM-treated tumors. These experiments have shown that the cell kinetic effect of TAM is different when MCF-7 cells are grown in vitro versus in vivo. In contrast to the in vitro data, the in vivo data indicate that the growth-inhibitory effect of TAM is not mediated through a perturbation of the cell cycle.     

三氧化二砷诱导人鼻咽低分化鳞癌裸鼠移植瘤细胞分化的研究

背景与目的:既往研究发现三氧化二砷(As2O3)可诱导白血病细胞的分化,但对实体瘤细胞的诱导分化作用报道甚少。本实验拟探讨As2O3对人鼻咽低分化鳞癌可移植瘤在BALB/C裸鼠体内生长的影响,重点观察其诱导鼻咽癌细胞分化的作用。方法:以人鼻咽低分化鳞癌鼠移植癌细胞株CSNE-1为研究对象,观察腹腔注射As2O3(5mg·kg-1·d-1连续10天后,每周给药3次,连续3周)对人鼻咽癌移植瘤在BALB/C裸鼠体内生长情况的影响。用光学显微镜和电子显微镜观察移植瘤细胞形态变化,用免疫组化法检测瘤组织中增殖细胞核抗原(PCNA)的表达情况。结果:腹腔注射As2O3后,人鼻咽癌BALB/C裸鼠移植瘤生长被抑制,抑瘤率为75.4%。同时,瘤组织中癌细胞密度减少,细胞皱缩,胞浆红染,瘤组织分化渐成熟,出现角化细胞和角化珠;间质结缔组织增多。透射电镜下肿瘤细胞出现成熟分化及明显角质化,细胞表面微小突起增多,细胞间桥粒增多并以桥粒互相连接,细胞核浆比例减少,胞浆中出现大量张力原纤维并围绕核周。癌细胞PCNA在阴性对照组呈高表达,PI(PCNA阳性细胞指数)为(95.2±5.0)%,而As2O3组癌细胞PCNA表达明显减少,PI为(53.6±7.0)%(P<0.001)。结论:As2O3抑制人鼻咽低分化鳞癌BALB/C裸鼠移植瘤的生长,这种抑制作用可能与其诱导癌细胞向成熟方向分化有关     

平阳霉素对大肠癌的抗肿瘤活性

目的：观察平阳霉素（ＰＹＭ）对大肠癌的活性。方法：应用小鼠结肠癌２６瘤株皮下移植和盲肠原位移植模型有裸鼠移植瘤模型,以１／９半数致死量（ＬＤ５０）等毒性剂量观察ＰＹＭ的抗瘤活性,并与丝裂霉素Ｃ（ＭＭＣ）和５－氟脲嘧啶（５－ＦＵ）进行比较。     

喉癌术前平阳霉素诱导性化疗的临床及基础研究

Objective： To evaluate the short-term effects of neoadjuvant chemotherapy with pingyangmycin （PYM） in the treatment of laryngeal squamous cell carcinoma. Methods： 24 cases of laryngeal squamous cell carcinoma were treated with PYM before the operation, and the surgeries were undergone within one week after neoadjuvant chemotherapy. PCNA, p53, Bcl-2 and CD44v6 were detected in the specimens of tumor, retreated tumor and normal tissue using immunohistochemical methods. Results： Apoptosis could be detected more often in specimens with tumor and retreated tumor after chemotherapy than that before. The expression of PCNA, p53, Bcl-2 and CD44v6 in tumor tissue after neoadjuvant chemotherapy with PYM was weaker than that before the chemotherapy. There was significant difference in the positive ratio of PCNA, p53, Bcl-2 and CD44v6 between retreated tumor and tumor. Conclusion： After neoadjuvant chemotherapy with PYM, a large number of tumor cells died. The amplification and metastasis of tumor were suppressed by neoadjuvant chemotherapy with PYM.     

Effect of inhibition of polyamine biosynthesis by DL-alpha-difluoromethylornithine on the growth and melanogenesis of B16 melanoma in vitro and in vivo

The objective of the present study was to investigate the effect of polyamine depletion by alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on the growth and differentiation of B16 melanoma cells grown in culture and also as solid tumors in mice. Polyamine depletion by DFMO (2.5 mM) resulted in a complete inhibition of cell growth in culture and a 90% inhibition of viability of melanoma cells as determined by clonogenic assay at the end of 7 days after DFMO treatment. These results indicate that polyamine depletion induced by DFMO is cytotoxic to B16 melanoma cells in culture. Furthermore a 2- to 5-fold increase in tyrosinase activity and 10-fold accumulation of melanine were observed in polyamine depleted cells compared to control cultures. These effects of DFMO could easily be reversed by the addition of putrescine simultaneously with DFMO. Administration of different doses of DFMO in drinking water to B16 melanoma tumor bearing mice also resulted in an increase in tyrosinase activity and a dose dependent inhibition (86-90%) of tumor growth. Although one cannot rule out the possibility of induction of differentiated phenotype as a result of antiproliferative activity of DFMO, the data presented indicate that the unique sensitivity of melanoma to DFMO may be due to a combination of cell growth inhibition and concomitant induction of differentiation upon polyamine depletion. The results of the present study indicate that polyamines play an important role in growth and differentiation of melanoma and also provide an example of inhibition of tumor cell growth by induction of cellular differentiation.     

Effects of progesterone on human endometrial carcinoma cells in vivo and in vitro

The effects of progesterone on the growth and differentiation of human endometrial adenocarcinoma cells (cell lines SNG-P and SNG-M derived from primary and metastatic tumors, respectively) were assessed in vitro and in vivo. Progesterone suppressed their growth and induced cell differentiation in vitro. The suppressive effect of progesterone was stronger in the primary tumor cells than in the metastatic ones. Progesterone produced morphologic changes such as multinucleation, multinucleolation, vacuolation, extensive Golgi apparatus, and papillary arrangement of cells. The cells were transplanted sc into nude BALB/c mice where they produced undifferentiated adenocarcinomas in untreated mice and well-differentiated adenocarcinomas in progesterone-treated ones. Progesterone reduced tumor growth and decreased transplantability in nude mice. This hormone produced no change in the distribution of the chromosome numbers or in the karyology.     

Induction of apoptosis in KB cells by pingyangmycin

Pingyangmycin (PYM; Bleomycin A(5)), an antitumour antibiotic is currently used during anticancer therapy. Previous experiments demonstrated that the therapeutic efficiency of PYM for treatment of malignant tumours is considered to be related to its ability to cause DNA strand breaks in vitro. However, very little is known about the interaction of PYM with the target cells, and it is still unclear how PYM enters the cells. In this study, cell death induced by PYM was studied in a human squamous cell carcinoma cell line (KB cells). In order to determine if cell death occurred by necrosis (reproductive cell death) or apoptosis (programmed cell death), KB cells were exposed to different concentrations of PYM and evaluated by biochemical and morphological criteria. Our results indicate that KB cells displayed an arrest in the G(2)-M phase of the cell cycle and became enlarged and polynucleated before dying at the low concentrations of PYM. In contrast, when cells were exposed to high concentrations of PYM, morphological changes identical to those usually associated with apoptosis were observed as well as internucleosomal digestion of genomic DNA. In conclusion, we demonstrate that PYM is able to induce two distinct modes of cell death depending on the doses of PYM.     

Physiological factors influencing radioantibody uptake: a study of four human colonic carcinomas.

We evaluated the accretion of 131I-labeled NP-4 anticarcinoembryonic antigen (CEA) into 4 size-matched human colonic carcinomas grown s.c. in nude mice. Antibody uptake for LS174T and GW-39 tumors was relatively high (19 to 23% ID/g on day 3), whereas moderate uptake was seen in the Moser tumor (7.5% on day 3) and low uptake was detected in the GS-2 tumor (1.8% on day 3). Blood clearance of radioantibody was twice as fast in mice with GS-2 tumors than in mice with GW-39, LS174T or Moser tumors. Seven physiological parameters that might influence radioantibody accretion were evaluated in order to better understand the differences in observed tumor targeting: vascular volume, blood flow rate, vascular permeability, tumor antigen content, serum antigen content and complexation of radioantibody, intratumoral antigen distribution, and intracellular antigen distribution. Although marked variability in vascular physiology, antigen content and antibody complexation of the 4 tumors grown in the same host and site existed, it was insufficient to explain the differences in antibody uptake. However, intra-tumoral distribution of antigen, and sub-cellular accessibility of antigen for radioantibody were important considerations. GS-2 tumors are well differentiated and have polarized cells. CEA in GS-2 is largely inaccessible to radioantibody; most of the antigen is located in the lumen of the glands or on the apical surface of gland cells and most of the antibody distributes to the stromal region on the basolateral surface. The low antibody targeting in GS-2 could therefore be explained by restricted intra-tumor accessibility of antibody. Scatchard analysis of NP-4 binding to Moser cells under non-internalizing and internalizing conditions revealed that 90% of the antigen is found within the cell, unavailable to bind with the NP-4 antibody, which is slow to internalize. In contrast, CEA in LS174T cells was almost entirely accessible. The reduced antibody targeting to Moser xenografts might therefore, be explained by restricted antibody accessibility at the cellular level.     

博来霉素A6和平阳霉素抗大肠癌实验研究

 本研究应用体外细胞毒试验,小鼠结肠癌移植瘤及裸鼠移植人大肠癌模型,观察博来霉素A6(A6)和平阳霉素(PYM)对大肠癌的疗效。结果发现2种药物在体外对人大肠癌细胞有较强的活性。在1/9LD50等毒性剂量下,A6和PYM对小鼠结肠癌26皮下和盲肠移植瘤的生长有明显的抑制作用,2次实验结果:对小鼠结肠癌皮下移植瘤,A6的抑瘤率分别为87％和88％,PYM为86％和91％,丝裂霉素C(MMC)为64％和25％,5—氟脲嘧啶(5—FU)为45％和27％;对盲肠移植瘤,A6抑瘤率分别为99％和97％,PYM为100％和97％,MMC为77％和25％,5—FU均为44％。同样A6和PYM对裸鼠移植的人结肠癌HT－29的生长也有较强的抑制作用,A6对肿瘤生长的抑制率为82％,PYM为78％,MMC为53％,5—FU为12％。表明A6和PYM对小鼠结肠癌的裸鼠移植的人结肠癌的生长抑制作用均明显较MMC和5—FU为强,A6的作用似略较PYM为强,但二者间无明显差别;在抑制肿瘤生长的同时,A6和PYM还使肿瘤的组织学发生变化,肿瘤坏死明显增加和核分裂数减少。在治疗剂量下A6和PYM对带瘤小鼠和荷瘤裸鼠的骨髓有核细胞数均无明显影响。     

Pingyangmycin downregulates the expression of EGFR and enhances the effects of cetuximab on esophageal cancer cells and the xenograft in athymic mice

Purpose  

As reported, epidermal growth factor receptor (EGFR) is over expressed in a variety of cancers including esophageal squamous cell carcinoma. Therefore, it becomes one of the potential targets for treating esophageal cancer. Pingyangmycin (PYM), a single A5 component of bleomycin, is currently used for the treatment of different types of cancers of epidermal origin, especially for head and neck cancers. In this report, the effect of PYM on EGFR expression in human esophageal cancer cells and the therapeutic efficacy of the combination of PYM and cetuximab on esophageal cancer xenograft were investigated.     

Antitumor Activities of IKP-104, a 4(1H)-Pyrizinone Derivative, on Cultured and Implanted Tumors

Antitumor activities of IKP-104, a 4(1 H )-pyrizinone derivative, were investigated with cultured tumor cell lines and implanted tumors in mice. IKP-104 inhibited the growth of cultured murine tumor cell lines (L1210 leukemia, Lewis lung carcinoma and B16 melanoma) and human tumor cell lines (K562 leukemia and HeLa cervical carcinoma). It also had antitumor effects on implanted murine ascitic tumors (L1210 leukemia and sarcoma 180) and a murine solid tumor (Lewis lung carcinoma). IKP-104 could be classified as a phase-dependent cytostatic drug based on the mode of growth inhibition of cultured B16 melanoma cells compared with those of several other antitumor agents. The effect of IKP-104 on the cell cycle traverse of cultured B16 melanoma cells was estimated by morphological and flow cytometric analyses. Cells accumulated in the mitotic phase, and abortive mitosis or polyploidy or multinucleation was induced from 6 h after exposure to IKP-104. Based on these results, IKP-104 is expected to be useful for the treatment of tumors, and its mode of action seemed to be similar to that of metaphase arrestants such as colchicine or vinca alkaloids.     

Antitumor activities of IKP-104, a 4(1H)-pyrizinone derivative, on cultured and implanted tumors

Antitumor activities of IKP-104, a 4(1H)-pyrizinone derivative, were investigated with cultured tumor cell lines and implanted tumors in mice. IKP-104 inhibited the growth of cultured murine tumor cell lines (L1210 leukemia, Lewis lung carcinoma and B16 melanoma) and human tumor cell lines (K562 leukemia and HeLa cervical carcinoma). It also had antitumor effects on implanted murine ascitic tumors (L1210 leukemia and sarcoma 180) and a murine solid tumor (Lewis lung carcinoma). IKP-104 could be classified as a phase-dependent cytostatic drug based on the mode of growth inhibition of cultured B16 melanoma cells compared with those of several other antitumor agents. The effect of IKP-104 on the cell cycle traverse of cultured B16 melanoma cells was estimated by morphological and flow cytometric analyses. Cells accumulated in the mitotic phase, and abortive mitosis or polyploidy or multinucleation was induced from 6 h after exposure to IKP-104. Based on these results, IKP-104 is expected to be useful for the treatment of tumors, and its mode of action seemed to be similar to that of metaphase arrestants such as colchicine or vinca alkaloids.     

肉桂酸诱导人肺腺癌细胞恶性表型逆转和分化作用

探讨肉桂酸对人肺腺癌A5 49细胞诱导分化作用及可能机制。方法　应用 2mmol/L终浓度肉桂酸作用于人肺腺癌A5 49细胞 ,观察体外生长增殖情况、双层软琼脂集落形成率并用流式细胞仪测定细胞周期及p5 3蛋白表达。结果　细胞体外生长速度减慢 ;双层软琼脂集落形成减少 ;细胞DNA合成能力降低 ;细胞周期出现向G1/G0期移行的特征性动力学改变 ;p5 3蛋白表达减少。结论　肉桂酸通过激活人肺腺癌细胞分化相基因表达启动细胞分化并达到恶性逆转 ,是一种有应用潜力的诱导分化剂。     

Restoration of impaired T cell functions in tumor-bearing mice by the administration of interleukin 1

The effect of in vivo administration of recombinant human interleukin 1 (IL-1) on T cell functions in tumor-bearing mice was studied using an in vitro assay system. The in vitro induction of trinitrophenyl (TNP)-specific cytotoxic T cell and proliferative T cells responses from spleen cells was impaired in X5563 plasmacytoma-bearing C3H/He mice. However, the administration of IL-1 alpha or IL-1 beta to tumor-bearing mice restored T cell functions in a dose-dependent manner. Antigen-presenting activities of spleen cells in tumor-bearing mice for T cell activation were not restored by the administration of IL-1. The activities of cytotoxic T cells and cytostatic T cells specific for X5563 cells were also enhanced by the administration of IL-1. Furthermore, in IL-1-treated mice, NK cell activity of spleen cells detected in terms of the killing of Yac-1 cells was also restored. In accordance with these results, the growth of X5563 cells was significantly inhibited and the lymphocytes from IL-1-treated mice specifically inhibited the growth of tumor cells. These results suggest that the in vivo administration of IL-1 restored the impaired T cell and NK cell functions in tumor-bearing mice and activated protective immunity against tumor cells. Thus, recombinant IL-1 can be applied for tumor immunotherapy.     

Effects of female sex steroid hormones on human endometrial adenocarcinoma transplanted into nude mice

In order to clarify the minute reactions of endometrial adenocarcinoma to sex steroid hormones, especially Progesterone (P), two kinds of in vivo models with differing sex steroid hormone receptor content were transplanted into nude mice. Subsequently, Estrogen (E2) and P were administered alone or together. Sequential histopathological changes, tumor growth curve and volume doubling time were investigated as compared to the castrated control group. Growth rate was increased by E2 administration compared to controls for the model positive for the E2 receptor (ER) and negative for the P receptor (PR), with PR production confirmed in 1 of 3 mice. P produced a slight inhibition of growth, and the inhibitory effect of P was additive with E2. The histological findings consisted of dense solid masses of cells in the tumor in mice treated with E2. The cells decreased in number and in some foci, the tubular glands were cystic in P-treated mice. The number of tumor cells in mice treated with E2 and P was less than in mice given P alone because of a secretive function seen in the appearance of PAS positive granules in the glandular epithelium and degenerative change such as swelling. With the tumor negative for both ER and PR, on the other hand, there was little difference in tumor cell growth between the treated and control groups, and no inhibitory effect was observed after administration of P or coadministration of E2 and P. Histologically, findings with increased nuclear division of tumor cells suggestive of tumor cell growth were obtained after administration of E2 alone and coadministration of E2 and P. E2 exhibited cell growth promoting action in endometrial adenocarcinoma, whether or not ER was present, and P appeared to inhibit tumor cell growth in the presence of ER even when PR was absent. In short, it was suggested that P acted by some mechanism independent of PR. After coadministration of E2 and P, tumor cells were affected in a direction toward functional differentiation in the ER positive tumor. The inhibitory effect of P was obviously additive with E2 in this tumor, while it was suggested that in ER negative tumor, E2 alone promotes tumor cell growth.     

TNF accelerates the S-phase of the cell cycle in tumor cells

The reduction of glucose supply induced the killing of tumor cells by tumor necrosis factor (TNF) in vivo and in vitro. In contrast, normal cell lines were resistant to TNF regardless of the presence or absence of glucose. Epidermal growth factor (EGF) did not exert a cytotoxic effect on tumor cells in the absence of glucose. Therefore, the killing mechanism of TNF under conditions of reduced glucose supply was investigated. Flow cytometry experiments and studies of kinetics revealed that the S-phase of the cell cycle was prolonged in the absence of glucose. After TNF treatment, the S-phase was found to be shortened and the rate of H-thymidine incorporation into DNA was increased, whereas EGF failed to exert such an effect. DNA synthesis and entry into mitosis are known to be regulated by cyclin A. In serum-starved tumor cells (HeLa) we have observed increased cyclin A synthesis within 10 hr, in parallel with enhancement of DNA synthesis and shortening of the S-phase after TNF treatment. We conclude that, under conditions of low glucose supply, TNF can assume the role of a growth factor in transformed cells.     

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.