Effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone (vitamin K3) treatment on human tumor cell growth in vitro. I. Synergism of combined vitamin C and K3 action

The effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone (vitamin K3) administered separately or in combination on the in vitro cultured human neoplastic cell lines MCF-7 (breast carcinoma), KB (oral epidermoid carcinoma), and AN3-CA (endometrial adenocarcinoma) have been examined. When given separately, vitamin C or K3 had a growth inhibiting action only at high concentrations (5.10(3) mumol/1 and 10(5) nmol/l, respectively). Combined administration of both vitamins demonstrated a synergistic inhibition of cell growth at 10 to 50 times lower concentrations. At this level separately given vitamins are not toxic. The sensitivity to this treatment was somewhat different in the three cell lines, being slightly higher for KB line. This tumor cell growth inhibitory effect was completely suppressed by the addition of catalase to the culture medium containing vitamins C and K3, suggesting an excessive production of hydrogen peroxide as being implied in mechanisms responsible for the above-mentioned effects.     

维生素C联合维生素K3抗肿瘤作用的研究进展

维生素C（vC）和维生素K3（VK3）在抗氧化、提高机体免疫力和止血等方面被广泛应用.20世纪40年代,人们发现它们也具有抗肿瘤作用.研究表明,VC与VK3联合给药比例为100：1时,具有最佳的协同抗肿瘤作用,并能大幅降低剂量,不会增加机体的不良反应.能够对乳腺癌、卵巢癌、前列腺癌、膀胱癌、白血病等多种肿瘤细胞产生杀伤作用.这种组合已被申请了专利并进行了Ⅰ/ⅡA期临床试验,试验结果表明其能延长晚期癌症患者的生存时间,不良反应轻,具有广阔的应用前景.文章就VC联合VK3的协同抗肿瘤作用进行综述.     

Non-toxic potentiation of cancer chemotherapy by combined C and K3 vitamin pre-treatment

The influence on the survival of ascitic liver tumor (TLT)-bearing mice of combined vitamins C and K3 administered before or after a single i.p. dose of 6 different cytotoxic drugs, all commonly used in human cancer therapy, was investigated. Combined i.p. administration of these vitamins produced a distinct chemotherapy-potentiating effect for all drugs examined, especially when injected before chemotherapy. This potentiating treatment did not increase the general and organ toxicity that accompanies cancer chemotherapy. The possible generation of peroxides followed by membrane lipid alteration, DNase activation and DNA destruction by combined vitamin C and K3 in catalase-deficient cancer cells might be involved in the mechanisms of this selective potentiation.     

A comprehensive review of vitamin K and vitamin K antagonists

For more than 60 years, vitamin K-dependent proteins have been known to play an important role in regulating blood coagulation. During recent years it has become clear, however, that vitamin K is also involved in other physiologic processes, including bone metabolism and vascular biology. Because the vitamin K requirement of bone and vessel wall is higher than that of the liver (where the clotting factors are produced) recommended daily allowance (RDA) values for K vitamins must be redefined. According to the new definition, a substantial part of the population is mildly deficient in vitamin K, and at later ages this deficiency may contribute to increased bone fracture risk, arterial calcification, and cardiovascular disease.     

Synergistic growth inhibition by acyclic retinoid and vitamin K2 in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. However, effective chemopreventive and chemotherapeutic agents for this cancer have not yet been developed. In clinical trials acyclic retinoid (ACR) and vitamin K(2) (VK(2)) decreased the recurrence rate of HCC. In the present study we examined the possible combined effects of ACR or another retinoid 9-cis retinoic acid (9cRA) plus VK(2) in the HuH7 human HCC cell line. We found that the combination of 1.0 microM ACR or 1.0 microM 9cRA plus 10 microM VK(2) synergistically inhibited the growth of HuH7 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VK(2) also acted synergistically to induce apoptosis in HuH7 cells. Treatment with VK(2) alone inhibited phosphorylation of the retinoid X receptor (RXR)alpha protein, which is regarded as a critical factor for liver carcinogenesis, through inhibition of Ras activation and extracellular signal-regulated kinase phosphorylation. Moreover, the inhibition of RXRalpha phosphorylation by VK(2) was enhanced when the cells were cotreated with ACR. The combination of retinoids plus VK(2) markedly increased both the retinoic acid receptor responsive element and retinoid X receptor responsive element promoter activities in HuH7 cells. Our results suggest that retinoids (especially ACR) and VK(2) cooperatively inhibit activation of the Ras/MAPK signaling pathway, subsequently inhibiting the phosphorylation of RXRalpha and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.     

细胞凋亡与细胞增殖协同作用的研究

在S期,砷剂在G1/S期,TNF-α或抗Fas抗体在G1/S期), PBLs不存在这种打击依赖性的差异.未经PHA刺激的PBLs对凋亡打击无应答,PBLs经PHA刺激后进入凋亡, 在外界打击作用下凋亡明显增加,在用咖啡因废除细胞周期检测点后对打击敏感性降低.拥挤或饥饿的PBLs同样对凋亡诱导无应答.结论:大多数细胞凋亡具有周期特异性,细胞周期改变可导致凋亡模式和时相的改变.细胞凋亡与增殖的协同作用是细胞周期和凋亡信号整合的重要机制.     

Signal transduction of vitamin K3 for pancreas cancer therapy

We characterized molecular mechanisms of vitamin K3 (VK3)-induced inhibition of proliferation to evaluate VK3 effectiveness in treating advanced pancreatic cancer. A novel endoscopic drug delivery system, ultrasound injection technique, was used to study local effects of VK3. VK3 inhibited pancreas cancer cell growth by rapid phosphorylation of growth factor receptor and cellular signal factors such as extracellular signal-regulated kinase. VK3 also activated apoptosis, and apoptosis inhibitor antagonized the apoptosis pathway without inhibiting cell growth. Thiol antioxidant treatment completely abrogated VK3-induced ERK but not JNK phosphorylation or inhibition of proliferation. Non-thiol antioxidant did not affect ERK phosphorylation or growth inhibitory actions. Arylation was considered the main mechanism of VK3-induced growth inhibition through ERK activation. VK3 may lead to favorable outcomes in the treatment of pancreatic tumors. Detection of ERK phosphorylation in tissue is important to predict VK3 effect. Endoscopic ultrasound-guided fine-needle injection may be beneficial for treating pancreatic cancer with VK3.     

Non-toxic sensitization of cancer chemotherapy by combined vitamin C and K3 pretreatment in a mouse tumor resistant to oncovin.

The effects of combined vitamin C and K3 i.p. injected 3 hours before i.p. administration of single dose of oncovin, to which the ascites liver tumor in mouse (T.L.T.) was completely resistant, were investigated. This pretreatment sensitized the tumor resistant to oncovin, whereas a separate pretreatment with vitamin C or K3 alone was without any effect. This tumor sensitization to the chemotherapy was completely suppressed by catalase pretreatment, thus indicating that hydrogen peroxide generation with subsequent oxidative stress and its consequences may be involved here. Since this sensitization was without any increased general and organ toxicity, its possible introduction into classical protocols of human cancer treatment would be without any supplementary risk.     

Synergistic cytotoxic effect of sulindac and pyrrolidine dithiocarbamate against ovarian cancer cells

Sulindac, a non-steroidal anti-inflammatory drug, suppresses carcinogenesis and inhibits growth of tumor cells. Pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, has been also identified as a potential anti-neoplastic agent. We hypothesized that combination of sulindac and PDTC could result in augmentation of cytotoxicity against ovarian cancer cells. The effect of sulindac and PDTC was examined on several ovarian cancer lines. Tumor cell viability was assessed using the MTT assay. Annexin-V/PI staining was used to detect apoptosis, cell cycle distribution was analyzed in FACS, and expression of cellular proteins was detected by western blotting. Incubation of OVA-14, OVP-10 and CAOV-1 ovarian cancer cells with sulindac and PDTC resulted in significantly greater inhibition of cell viability compared to either compound alone. In a model of OVA-14 cells it was evident that this effect was not related to the expression of COX enzymes since both active (sulindac sulfide) and inactive (sulindac) in vitro compounds affected the growth of tumor cells to a similar extent and synergized in cytotoxicity with PDTC. Combination of sulindac and PDTC lead to G0 arrest and massive apoptosis in co-treated cultures. Western blotting analysis argued for induction of the mitochondrial apoptotic pathway. These data demonstrate the synergistic cytotoxic effect of sulindac and PDTC on ovarian cancer cells through apoptosis and cell cycle arrest and prompt to test the efficacy of this combination in animal models.     

Radiosensitizing and cytotoxic properties of mitomycin C in a C3H mouse mammary carcinoma in vivo

The radiosensitizing and cytotoxic properties of Mitomycin C (MMC) was investigated in vivo using regrowth delay and tumor control assays. MMC significantly enhanced the radiation-induced growth delay when administered 15 min before irradiation; the slope of the dose response curve significantly increased and corresponded to a Dose Modifying Factor (DMF) of 1.9 (1.5-2.3; p less than 0.001). When MMC was given 4 hr after irradiation, the additional regrowth delay resulted in a parallel shift of the dose response curve, and MMC was not significantly dose modifying (DMF 1.3 (0.9-1.3); p less than 0.05). From isobologram analysis it was found that the preirradiation MMC schedule resulted in supra-additive responses, whereas MMC given after irradiation had an additive effect. The enhancement of radiation-induced tumor control was similarly found to peak when MMC was given 6 hr to 15 min prior to irradiation. At these intervals, the observed TCD50 for the combined treatments relative to radiation alone corresponded to Enhancement Ratios of 1.27 and 1.29, respectively (p less than 0.001). Longer intervals between the modalities reduced the enhancement, but the combined treatments were still significantly better than radiation alone (ER 1.12, 1.16 and 1.17; p less than 0.001). The significant enhancement of tumor control correlated with a substantial drug-induced cytotoxic effect toward hypoxic tumor cells, as determined by clamped TCD50 experiments. A single dose of MMC (3 mg/kg) was found to kill up to 97% of all hypoxic tumor cells.     

Synergistic cytotoxic and antitumor effects of recombinant human tumor necrosis factor and hyperthermia

A synergistic increase in the cytotoxic effects of recombinant human tumor necrosis factor (rH-TNF) and hyperthermia was demonstrated both in vitro and in vivo. The cytotoxicity of rH-TNF against L-M cells in incubation for 12 h at 38.5 and 40 degrees C based on the concentration necessary for 50% cytotoxicity was, respectively, 125 and more than 500 times as high as in similar incubation at 37 degrees C. As observed 18 days after implantation of Meth-A fibrosarcoma cells in mice, single i.v. administration of rH-TNF at 1000 units/mouse resulted in complete cures in five mice when performed in combination with hyperthermia (40 degrees C), whereas rH-TNF alone in the same dose resulted in 27.1% inhibition of tumor growth and hyperthermia alone had no appreciable effect on tumor growth. The i.v. administration of rH-TNF three times at 100 or 300 units/mouse together with hyperthermia (40 degrees C) resulted in 41.2 and 89.0% tumor growth inhibition, respectively; similar administration without hyperthermia appeared to have little or no appreciable effect on tumor growth. The results suggest that combination therapy including rH-TNF and hyperthermia may be of value in the treatment of malignancy in human patients.     

New insights on vitamin K

Vitamin K catalyzes the post-translational carboxylation of coagulation proteins C, S, and factors II, VII, XI, and X. Detection of the noncarboxylated forms allows an indirect and specific measure of the vitamin K deficiency found in early, classic, and late hemorrhagic disease of the newborn (HDN), malabsorption syndromes, and drug related (warfarin, anticonvulsants, and antibiotics) states. Idiopathic late HDN (CNS bleeding) occurs in exclusively breast-fed infants and is prevented by appropriate parenteral and oral vitamin K prophylaxis given at birth. All newborn infants and older infants with malabsorption syndromes should receive prophylactic vitamin K.     

Synergistic cytotoxic effect between serine-threonine phosphatase inhibitors and 5-fluorouracil: a novel concept for modulation of cytotoxic effect

PURPOSE: The present study was undertaken to look for an agent or agents able to modulate the cytotoxic effect of 5-fluorouracil (FUra) and to investigate the role of serine-threonine phosphatase inhibitors on the cytotoxic effect of FUra. METHODS: The cytotoxicities of FUra and protein phosphatase inhibitors (PPIs) were evaluated by two different methods: a clonogenic assay and a proliferation assay. In the clonogenic assay, cancer cells were treated with various concentration of FUra with or without PPIs for 72 h. The drug-containing medium was replaced by fresh medium, the cultures incubated for an additional 10 days, and the colonies enumerated. In the proliferation assay the cells were treated with FUra alone or in combination with PPIs for 96 h and cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on the uptake of the tetrazolium dye. Thymidine kinase (TK) activity was determined based on the catalytic phosphorylation of [(3)H]d-thymidine to [(3)H]dTMP. Incorporation of FUra into DNA and RNA was determined by treating the cells with [2-(14)C]fluorouracil for 72 h and measuring the radioactivity in the isolated DNA and RNA fractions. RESULTS: The serine-threonine phosphatase inhibitors caliculin A (CAL), okadaic acid (OA) and microcystin-LR (MCLR) dose-dependently inhibited the growth of Clone 20 and Clone 5 cells of Colon 26 murine colon adenocarcinoma cells, human cervical cancer HeLa cells, human gastric cancer MKN 7 cells, and murine sarcoma S-180 cells in vitro. Among the compounds tested, MCLR at non-toxic concentrations was found to increase FUra incorporation into RNA and DNA in Clone 20 cells by 60% and 127%, respectively, to increase TK activity alone (twofold) as well as in combination with FUra (threefold), and to potentiate the cytotoxicity of FUra synergistically and cytospecifically in vitro. The cytotoxicity of FUra alone or in combination with MCLR, but not that of PPIs alone, was abrogated almost completely by exogenous thymidine (dThd), suggesting that inhibition of thymidylate synthetase (TS) is the growth-limiting event in the cytotoxic action of FUra even in combination with MCLR. CONCLUSIONS: The findings presented here suggest that MCLR synergistically and cytospecifically potentiates the antitumor activity of FUra with substantial improvement in the therapeutic index of FUra via enhancement of both DNA- and RNA-directed cytotoxicity.     

免疫毒素与顺铂协同杀伤肿瘤细胞机制的探讨

背景与目的:免疫毒素HELβ-PE38KDEL(HeregulinEGFlikedomainβ1-PsedomonasAeurginosa38KDEL,本文简称ITH)已经被证实是一种具有特异性杀伤erbB2、3、4阳性乳腺癌细胞的新的生物学制剂,然而它与化疗药物的联合作用效果目前尚未有报道。本文探讨ITH与化疗药物顺铂(DDP)的协同抗肿瘤作用。方法:采用AnnexinV结合实验和Westernblot检测乳腺癌细胞MDA-MB-453、2LMP和胃癌细胞N87分别在ITH和DDP单独及联合用药前后的细胞凋亡变化。结果:在高表达erbB-2、3、4的MDA-MB-453和N87中,联合用药组和单药组相比,凋亡细胞成倍增加(P<0.01);而在低表达erbB-2、3、4的2LMP,联合用药组和单药组相比,凋亡细胞无明显增加(P>0.05)。MDA-MB-453和N87细胞在联合用药组中PARP、caspase-3降解增加,bcl-2、mp53过度表达受抑制;而2LMP细胞中PARP、caspase-3降解无增加,bcl-2、mp53过度表达未受抑制。结论:ITH和DDP联合后可促进高表达erbB-2、3、4的乳腺癌细胞凋亡,这可能是两者协同作用的机制之一。     

Synergistic cytotoxic actions of cisplatin and liposomal valinomycin on human ovarian carcinoma cells

Summary We have previously shown that the toxicity of valinomycin (VM), a membrane-active agent with antineo-plastic activity, can be dramatically reduced with no loss of the antitumor efficacy of the drug by incorporating it into liposomes. In the present study, we investigated the interaction betweencis-diamminedichloroplatinum(II) (CDDP) and VM in terms of in vitro cytotoxicity to human ovarian tumor cells. Using the MTT assay and analyzing the data using the median-effect principle, we showed that synergistic cytotoxic interactions exist between CDDP and VM in their liposomal form. The degree of cytotoxic synergism was influenced by the duration of drug exposure and the dose ratio. The cellular accumulation of platinum by ovarian cells at 37°C was slightly higher after exposure to VM as compared with controls; however, it is not clear that this accounts for the cytotoxic synergism. These results suggest that the combination of liposomal VM and CDDP may have merit as a form of localized drug delivery for the treatment of ovarian cancer disseminated within the peritoneal space.Abbreviations CDDP cisplatin,cis-diamminedichloroplatinum(II) - VM valinomycin - MLV-VM liposomal valinomycin - MTT 3-(4,5-dimethyl thiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (thiazolyl blue) - alpha-MEM alpha minimal essential medium - CHO Chinese hamster ovary - IC₅₀ concentration causing 50% inhibition of cell growth - IC₁₀ concentration causing 10% inhibition of cell growth - SF surviving fraction - fa fraction affected - CI combination index     

Synergistic effects of co-expression of the Th1 cytokines il-2 and IFNγ on generation of murine tumor-reactive cytotoxic cells

While the effect of cytokines on the generation of tumor-reactive cytotoxic cells has been a topic of active investigation, the effect of physiological cytokine combinations has not been determined. We have investigated the effect of co-expression of IL-2 and IFN-γ on the generation of cytotoxic cells against the murine line 1 tumor in vivo. These cytokines were selected because they are normally produced in concert by a subset of T-helper cells called T-helper 1 (Th1). We transfected the line 1 murine carcinoma with cDNA for IL-2 and IFN-γ, alone or combined. IFN-γ alone does not elicit rejection of the transfectant, but IL-2 increases the tumorigenic dose by 10,000-fold above the parental cells. Co-expression of IFN-γ and IL-2 increases this rejection to at least 100,000-fold above parental line 1. Unlike IL-2 transfectants, tumor cells expressing both IFN-γ and IL-2 can also elicit rejection of admixed parental tumor cells. Finally, the IFN-γ/IL-2 transfectants are more effective at generating memory cells that are cytolytic for the parental tumor. Our results show that synergistic interactions of Th1 cytokines can remarkably enhance the cytotoxic response to tumors. © 1995 Wiley-Liss, Inc.     

维生素K2与骨质疏松症的研究进展

骨质疏松症是一种骨量降低,骨组织微结构破坏,以骨脆性增加,骨强度下降,骨折风险增加为特征的全身骨骼系统疾病。近年来,随着老龄化人数的增加,骨质疏松症已经严重威胁了中老年人身体健康,成为人类社会共同关注的重要健康问题。据一项全国性流行病学调查显示,50岁以上男性骨质疏松症患病率为14．4％,女性升高到20．7％,60岁以上人群的患病率进一步升高。     

Augmentation of cytotoxic drug action and X-irradiation by antibodies.

The effect of an antiserum containing antibodies against cell surface components of PyBHK cells on the action of certain anticancer agents has been studied using a colony formation inhibition assay. The effects of x-rays, chlorambucil, CCNU and possibly ICRF 159 are augmented by the antiserum whereas methotrexate and vinblastine are not.     

Synergistic action of fMLP-boanmycin combination on the growth of mouse colon carcinoma and its action mechanisms

Objective: The inhibitory action of fMLP-boanmycin (BAM) combination on the growth of mouse colon carcinoma and its action mechanisms were observed in order to provide experimental proof for probing novel regimen of chemotactic modulation in combination with chemotherapy in the treatment of cancer.Methods: Cytotoxicity of BAM-fMLP combination to tumor cells was determined by MTT assay in vitro. Antitumor activity of BAM-fMLP combination was assessed in mice subcutaneously transplanted colon carcinoma 26. The amount of superoxide anion (O2·-) released from fMLP stimulated macrophages was determined by NBT assay. The amount of nitric oxide (NO) was indirectly determined by Griess method. Results: BAM-fMLP combination had no synergistic effect on tumor cells(CDI>0.85), but BAM at the doses of 10μg/ml, 30μg/ml and 100μg/ml in combination with fMLP at the concentration 20μg / ml exhibited synergistic effect on tumor cells in the presence of macrophages(CDI<0.75), fMLP inhibited the growth of colon carcinoma 26 by 50.0% when it at dose of 1 mg/mouse was administered peritumorally. BAM (1 mg/kg, intraperitoneally, three times) alone and BAM - fMLP combination inhibited the growth of colon carcinoma 26 by 38.6% and 78.4%, respectively (CDI=0.71) on day 12. The amount of O2·- released from fMLP 4.6×10-77 mol/L (0.2μg/ml) stimulated macrophages which were treated by BAM in vitro increased significantly(P<0.01), fMLP 2.3×10-6 mol/L (1μg/ml) could not stimulate macrophages to release NO, but may stimulate macrophages treated with BAM 10μg/ml and 100μg/ml to release NO significantly(P<0.01). Conclusion: The inhibitory action of fMLP-boanmycin combination on the growth of mouse colon carcinoma have synergism, which may associate with the increase of O2·- and NO released by macrophages. Chemotactic modulation in combination with chemotherapy may be a novel regimen in the treatment of cancer.