Leptin promotes the growth of Colon 38 cancer cells and interferes with the cytotoxic effect of fluorouracil in vitro

INTRODUCTION: Epidemiological studies underline the fact that obesity represents a significant risk factor for the development of several cancers, one of which is cancer of the colon. Moreover, multiple recent data indicate that some adipose tissue-derived hormones may influence the growth of malignant cells. Leptin, the product of the ob gene, is one of these. However, the evidence from research is still contradictory regarding the role of leptin in colon cancer. The aim of our study was to examine the direct effect of leptin at various concentrations (from 10(-5) to 10(-12) M) when applied alone or jointly with fluorouracil (the classical cytotoxic drug for colon cancer) at two concentrations (0.25 mg/ml and 2.5 mg/ml) on the growth of murine Colon 38 cancer cells in vitro. MATERIAL AND METHODS: Colon 38 cancer cells were preincubated in RPMI 1640 medium supplemented with foetal calf serum for 24 hours. The cells were then cultured for a further 72 hours in the presence of various concentrations of the substances under examination, applied either alone or jointly. The growth of the Colon 38 cell line was assessed by a colorimetric kit based on the modified Mosmann method. RESULTS: We found that leptin increased the growth of murine Colon 38 cancer at concentrations of 10(-6), 10(-7) M and 10(-10), 10(-11), 10(-12) M. Its stimulatory effect was fairly slight, with an increase in cancer growth of 5% to 15% as compared to controls. As we expected, fluorouracil at both the concentrations examined inhibited the growth of Colon 38 cancer maximally up to 28% (2.5 mg/ml) and 34% (0.25 mg/ml) of controls, with a stronger effect obtained from higher doses. Leptin did not modulate the cytotoxic effect of fluorouracil applied at the higher concentration (2.5 mg/ml) but, unexpectedly, at concentrations of 1(-9) and 10(-10) M it heightened the cytotoxic effect of fluorouracil given at a lower concentration (0.25 mg/ml). CONCLUSIONS: These data indicate that leptin is involved in the regulation of colon cancer growth and it may even heighten the cytotoxic effect of fluorouracil.     

The combined effects of proglumide and fluorouracil on the growth of murine Colon 38 cancer cells in vitro

The role of gastrins and their receptors in the pathogenesis of colon cancer has been discussed for many years but it still remains unresolved. Although fluorouracil (FU) remains to be reference chemotherapy for colon cancer, its efficacy is unsatisfactory. Recently, we have shown a synergistic effect of proglumide (a non-selective blocker of cholecystokinin-gastrin receptors) applied together with FU, on the proliferation and apoptosis of transplantable Colon 38 cancer cells in vivo. The aim of this study was to examine direct effects of proglumide (10(-5)-10(-10) M) applied either alone or together with FU (0.25, 2.5 and 25 microg/ml) on the proliferation of murine Colon 38 cancer cells in vitro. Cell proliferation was assessed by the modified colorimetric Mosmann method. Proglumide inhibited the proliferation of Colon 38 cells at the concentrations of 10(-6), 10(-8) and 10(-10) M. FU inhibited the proliferation of cancer cells in all studied concentrations, exerting the most profound antiproliferative effect at the concentrations of 2.5 and 25 microg/ml. Thus, the former concentration was chosen to study its interactions with proglumide. Proglumide applied together with FU exerted a synergistic effect on the inhibition of proliferation of Colon 38 cells at 10(-8), 10(-9), 10(-10) M concentrations. The most profound inhibitory effect was observed in the group incubated with FU and 10(-10) M of proglumide. The obtained results indicate a possibility of new therapeutic options for colon cancer, but further studies are needed to elucidate, if the synergistic effect of FU and proglumide occurs also in the colon cancer in humans.     

Estrone and progesterone inhibit the growth of murine MC38 colon cancer line

The unsatisfactory effectiveness of reference chemotherapy in colon cancer (fluorouracil – FU) results in continuous search for agents, which could enhance the action of FU. Some epidemiological data such as a decreased risk of colorectal cancer among menopausal women receiving hormonal replacement therapy indicate the role of female sex hormones in the pathogenesis of this disease.The aim of this study was to examine the direct effects of various concentrations of estrone and progesterone (10^?4 to 10^?12 M) applied alone or together with FU on the growth of murine MC38 colon cancer in vitro.Estrone inhibited MC38 cancer growth in a wide range of concentrations (10^?12 to 10^?4 M) with similar potency and at some concentrations (10^?6 and 10^?4 M) augmented also the cytotoxic action of FU. Progesterone induced MC38 cancer growth inhibition at high concentrations (10^?5 to 10^?4 M) in dose- and time-dependent manner but it did not intensify antineoplastic effect of FU. A weak inhibitory effect of progesterone was also observed for lower concentrations (10^?5 to 10^?10 M) in long lasting cultures (72 h).The results indicate that estrone and progesterone inhibit the MC38 cancer growth and that estrone increases also the cytotoxic effect of FU, what confirms the role of female sex steroids in modulation of colon cancer growth.     

VIP-ellipticine derivatives inhibit the growth of breast cancer cells

The effects of vasoactive intestinal peptide (VIP)-ellipticine (E) derivatives were investigated on breast cancer cells. VIP-ALALA-E and VIP-LALA-E inhibited 125I-VIP binding to MCF-7 cells with an IC(50) values of 1 and 0.2 microM respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in MCF-7 cells with ED(50) values of 1 and 0.1 microM. VIP-LALA-E caused increased c-fos mRNA in MCF-7 cells. Radiolabeled VIP-LALA-E was internalized at 37 degrees C and delivered the cytotoxic E into MCF-7 cells. VIP-LALA-E inhibited the clonal growth of MCF-7 cells, decreased cell viability based on trypan blue exclusion and reduced 35S-methionine uptake. These results indicate that VIP-E derivatives function as breast cancer VPAC(1) receptor agonists which inhibit MCF-7 cellular viability.     

Oestradiol treatment increases the sensitivity of MCF-7 cells for the growth stimulatory effect of IGF-I

MCF-7 cells were grown in serum free medium (Dulbecco MEM without phenol red, supplemented with Costar SF-1 without insulin). Insulin was added as required and gave dose dependent growth stimulation at concentrations between 5 and 10,000 nM. Identical growth response curves were obtained for thymidine uptake and cell number. Oestradiol and insulin-like growth factor I (IGF-I) added individually both gave a dose dependent stimulation of cell growth in serum free medium containing 50 nM insulin. The growth stimulatory effect of oestradiol was to a large extent inhibited with suramine, a general inhibitor of growth factors, indicating that the effect of oestradiol was mediated through stimulating autocrine secretion of a growth factor.

To investigate a possible link between the effects of oestradiol and IGF-I, a specific IGF-I receptor antibody (IR-3), 10 μg/ml was used. These experiments were carried out with 2.5 nM insulin in the medium, a concentration at which insulin had no growth stimulatory effect. Stimulation was carried out for 18 h before assay of thymidine uptake. The effect of oestradiol was not significantly reduced by IR-3, indicating that IGF-I was not an autocrine mediator of oestradiol stimulation of cell growth under these conditions, whereas IR-3 extensively reduced growth stimulation by IGF-I. On long term stimulation (5 days) oestradiol had a marked stimulatory effect on cell number and IR-3 almost totally abrogated this effect. When oestradiol (1 nM) and IFG-I (2.5 nM) were added together, the combined effect on thymidine incorporation and cell number was significantly greater than additive. This synergistic effect on the IGF-I growth response was totally abolished by the IGF-I receptor antibody. The results suggest a cooperative interaction of oestradiol and IGF-I. It is concluded that growth stimulation of MCF-7 cells by long term treatment with oestradiol may be mediated through autocrine secretion of IGF-I.

The effect of short term stimulation of thymidine incorporation suggest that the growth response of oestradiol is more complex, and indicate that a cooperative interaction with IGF-I is involved, which is unrelated to stimulated autocrine secretion.     

Dexamethasone and zinc in combination inhibit the anchorage-independent growth of S-91 Cloudman murine melanoma

Zinc inhibited the colony formation of Cloudman S-91 murine melanoma cells in a dose dependent manner with an ID50 of 3.4 ug/ml. Total inhibition of the melanoma colony-forming units occurred at a zinc concentration of 4.42 ug/ml. In the presence of dexamethasone the ID50 for zinc inhibition was reduced by 49% and total inhibition of anchorage-independent growth occurred at the achievable in vivo zinc concentration of 3.0 ug/ml. Dexamethasone and zinc in combination effected a greater than additive inhibition of the murine melanoma colony-forming units. Statistical evaluation of these results showed that zinc and dexamethasone interacted synergistically to inhibit the formation of murine melanoma colonies.     

Environmental magnetic fields inhibit the antiproliferative action of tamoxifen and melatonin in a human breast cancer cell line

We have previously reported that environmental-level magnetic fields (1.2 μT [12 milligauss], 60 Hz) block the growth inhibition of the hormone melatonin (10⁻⁹ M) on MCF-7 human breast cancer cells in vitro. We now report that the same 1.2 μT, 60 Hz magnetic fields significantly block the growth inhibitory action of pharmacological levels of tamoxifen (10⁻⁷ M). In biophysical studies we have taken advantage of Faraday's Law of Current Induction and tested whether the 1.2 μT magnetic field or the associated induced electric field is responsible for this field effect on melatonin and tamoxifen. We observe that the magnetic field component is associated with the field blocking effect on melatonin and tamoxifen function. To our knowledge the tamoxifen studies represent the first experimental evidence for an environmental-level magnetic field modification of drug interaction with human breast cancer cells. Together, these findings provide support to the theory that environmental-level magnetic fields can act to modify the action of a drug or hormone on regulation of cell proliferation. Melatonin and tamoxifen may act through different biological pathways to down-regulate cell growth, and further studies are required to identify a specific biological site of interaction for the 1.2 μT magnetic field. Bioelectromagnetics 18:555–562, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Recombinant human Erythropoietin enhanced the cytotoxic effects of tamoxifen toward the spheroid MCF-7 breast cancer cells

Erythropoietin (EPO) is widely used to treat anemia in patients undergoing chemotherapy for cancers. The main objective of this study was to investigate the effect of rHuEPO on the response of spheroid breast cancer, MCF-7, cells to tamoxifen treatment. The MCF-7 spheroids were treated with 10 mg/mL tamoxifen in combination with either 0, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The viability of the MCF-7 cells was determined using the annexin-V, cell cycle, caspases activation and acridine orange/propidium iodide staining. rHuEPO-tamoxifen combination significantly (p greater than 0.05) increased the number of spheroid MCF-7 cells entering early apoptotic phase after 12 h and late apoptotic phase after 24 h of treatment; primarily the result of the antiproliferative effect tamoxifen. Tamoxifen alone significantly (p < 0.05) increased the caspase-3 and −9 activities in the spheroid MCF-7 cells by 200 to 550% of the control. Combination rHuEPO and tamoxifen produced much lesser effect on the caspase-8 activity. The rHuEPO in the combination treatment had concentration-dependently caused decrease in the caspase activities. rHuEPO-tamoxifen combination markedly increased MCF-7 cells entering the SubG0/G1 phase of the cell cycle by more than 500% of the control, while decreasing those entering the G2 + M and S phases by 50%. After 72 h, the combination treatment produced greater (p < 0.05) change in the SubG0/G1 phase than tamoxifen treatment alone. Morphologically, spheroid MCF-7 cells subjected to combination rHuEPO-tamoxifen treatment showed nuclear condensation and margination, cytoplasmic blebbing, necrosis, and early and late apoptosis. Thus, the study showed that rHuEPO-tamoxifen combination induced apoptosis in the spheroid MCF-7 cells. The apoptotic effect of the rHuEPO-tamoxifen combination treatment on the MCF-7 cells was greater than that produced by tamoxifen alone. The rHuEPO-tamoxifen treatment enhanced the caspase-independent apoptotic effects of tamoxifen on the spheroid MCF-7 cells.     

Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens

The antiproliferative effect of antiestrogens in breast cancer is believed to be entirely due to the inhibition of estrogen induced growth. We show here that non-steroidal antiestrogens inhibit the growth of the human breast cancer MCF7 cells in the complete absence of estrogens (phenol-red-free medium) when cell proliferation is stimulated by insulin or epidermal growth factor. This non-antiestrogenic effect of antiestrogens is, however, mediated by accessible estrogen receptor sites, as it is not observed in receptor negative hormone-independent breast cancers, and is rescued by estradiol but not by insulin. We conclude that antiestrogens inhibit cell proliferation by inhibiting growth factor action as well as estrogen action and that in both cases, accessible estrogen receptors are required.     

Lnc-DC promotes estrogen independent growth and tamoxifen resistance in breast cancer

Selective estrogen receptor modulators (SERMs) such as tamoxifen have proven to be effective in the treatment of estrogen receptor (ER) positive breast cancer. However, a major obstacle for such endocrine therapy is estrogen independent growth, leading to resistance, and the underlying mechanism is not fully understood. The purpose of this study was to determine whether long non-coding RNAs (lncRNAs) are involved in regulation of estrogen independent growth and tamoxifen resistance in ER positive breast cancer. Using a CRISPR/Cas9-based SAM (synergistic activation mediator) library against a focus group of lncRNAs, we identify Lnc-DC as a candidate lncRNA. Further analysis suggests that Lnc-DC is able to reduce tamoxifen-induced apoptosis by upregulation of anti-apoptotic genes such as Bcl2 and Bcl-xL. Furthermore, Lnc-DC activates STAT3 by phosphorylation (pSTAT3^Y705), and the activated STAT3 subsequently induces expression of cytokines which in turn activate STAT3, forming an autocrine loop. Clinically, upregulation of Lnc-DC is associated with poor prognosis. In particular, analysis of a tamoxifen-treated patient cohort indicates that Lnc-DC expression can predict the response to tamoxifen. Together, this study demonstrates a previously uncharacterized function of Lnc-DC/STAT3/cytokine axis in estrogen independent growth and tamoxifen resistance, and Lnc-DC may serve as a potential predictor for tamoxifen response.Subject terms: Breast cancer, Non-coding RNAs     

The effect of leucovorin on the therapeutic index of fluorouracil in cancer patients

Fluorouracil has been in clinical use as an anticancer drug for 30 years. Although this drug has a broad spectrum of anticancer activity, including significant activity against the common solid tumors of the gastrointestinal system, only a minority of patients treated with fluorouracil experience an objective response to therapy. Furthermore, in randomized clinical trials completed to date, it has not been possible to demonstrate that fluorouracil therapy significantly prolongs the life span of patients with advanced cancer. Recent laboratory studies have indicated that leucovorin can enhance the cytotoxicity of fluorouracil in vitro, evidently by enhancing inhibition of the key enzyme, thymidylate synthetase, by the fluorouracil metabolite, FdUMP (fluorodeoxyuridine monophosphate; a stable inactive FdUMP-reduced folate-thymidylate synthetase complex is formed). Pilot, uncontrolled studies of leucovorin-fluorouracil combinations have suggested that leucovorin may significantly increase both the clinical efficacy and the clinical toxicity of fluorouracil in cancer patients. These findings have led to the initiation of several randomized, controlled studies of leucovorin plus fluorouracil versus fluorouracil alone in the treatment of patients with advanced colorectal cancer. Three of these studies have recently completed patient accrual, and the preliminary results of each of the three studies indicate that leucovorin-fluorouracil combinations will have a better therapeutic index than fluorouracil used alone in this disease. Further follow-up of these studies will be needed to determine whether leucovorin-fluorouracil combination therapy will prolong the life span of patients with colorectal cancer.     

The theaflavin monomers inhibit the cancer cells growth in vitro

The biological effects of tea and tea constituents havebeen studied and reviewed in many publications. Teaand its components have been demonstrated to inhibitchemically induced carcinogenesis in animal models,including cancers of the skin, lung, esophagus…     

Studies on tamoxifen encapsulated in lipid vesicles: effect on the growth of human breast cancer MCF-7 cells

Tamoxifen is a nonsteroidal estrogen-receptor modulator widely used in the treatment of breast cancer. Apoptosis has been reported to be a major mechanism for its antitumor effect. In the current studies, an endeavor was made to investigate the efficacy of vesicularly encapsulated tamoxifen on human breast cancer MCF-7 cells. Phospholipid-based vesicular systems viz. conventional liposomes and elastic-membrane liposomes were employed to encapsulate the drug. The MTT colorimetric assay was used to determine the efficacy of the tested formulations. The results demonstrated composition-dependent strong inhibition in the viability of MCF-7 cells with encapsulated tamoxifen vis-à-vis free drug. The encouraging findings from the current work construe immense potential of the lipid-based vesicular systems in the treatment of breast cancer.     

Bombesin marine toxin conjugates inhibit the growth of lung cancer cells

Hemiasterlin (Hem) and dolastatin (Dol) are marine natural products which are cytotoxic for cancer cells. Hem, a tripeptide, and Dol, a hexapeptide, were conjugated with linkers (L) to the universal BB agonist DPhe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2(BA1) and the effects of the Hem-BB and Dol-BB conjugates investigated on NCI-H1299 lung cancer cells. Hem-LA-BA1 and Hem-LB-BA1 inhibited specific (125I-Tyr4)BB binding to NCI-H1299 cells, which have BB2 receptors (R), with IC50 values of 15 and 25 nM, respectively. Addition of Hem-LA-BA1 and Hem-LB-BA1 to Fura-2 AM loaded cells containing BB2R, caused elevated cytosolic Ca2+. In a growth assay, Hem-LA-BA1 and Hem-LB-BA1 inhibited the proliferation of NCI-H1299 cells. Dol-succinamide (Dols)-LD-BA1 and Dols-LE-BA1 bound with high affinity to NCI-H1299 cells and elevated cytosolic Ca2+, but did not inhibit the proliferation of NCI-H1299 cells. Also, Hem-LA-BA1 inhibited 125I-DTyr-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 (BA2) binding to Balb/3T3 cells transfected with BB1R or BB2R as well as with BRS-3 with IC50 values of 130, 8, and 540 nM, respectively. These results show that Hem-BB conjugates are cytotoxic for cancer cells containing BB2R.     

Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates

Murine peritoneal macrophages were isolated and their ability to restrict growth of a virulent Mycobacterium tuberculosis in response to IFN-gamma was assessed in various conditions. Doses of IFN-gamma ranging from 10 to 100 U stimulated high levels of antimycobacterial activity, as seen by inhibition of growth. Addition of catalase, superoxide dismutase, and other scavengers of reactive oxygen species before infection failed to abrogate this restriction of growth, suggestive of a lack of involvement of reactive oxygen species in this phenomenon. Addition of arginase before infection inhibited the bacteriostatic ability of IFN-gamma-pulsed macrophages as did addition of NG-monomethyl L-arginine, an inhibitor of the synthesis of inorganic nitrogen oxide. In both cases, this inhibition was reversed by adding excess L-arginine in the medium. Moreover, nitrite production in macrophages was correlated with their ability to restrict tubercle bacilli growth. These results imply that nitric oxide or another inorganic nitrogen oxide is an important effector molecule in restricting growth of M. tuberculosis in IFN-gamma-pulsed murine macrophages.     

Sigma ligands inhibit the growth of small cell lung cancer cells

The effects of sigma ligands on small cell lung cancer (SCLC) cells were investigated. 125I-N-(2-(piperidino)ethyl)-2-iodobenazmide (2-IBP) bound with high affinity to SCLC cell line NCI-H209 and NCI-N417. Specific 125I-2-IBP binding was inhibited with high affinity by ifendipine, haloperidol, (2-piperidinyl-aminoethyl)-4-iodobenzamide (IPAB) and 1,3-ditolylguanidine (DTG) with IC50 values of 3, 10, 15 and 90 nM respectively. In vitro, 10 microM 2-IBP, haloperidol or IPAB inhibited NCI-N417 proliferation using a MTT or clonogenic assay. In vivo, 4 mg/kg IPAB or 2-IBP inhibited NCI-N417 xenograft proliferation. 125I-2-IBP localized to the SCLC tumors after subcutaneous injection. These results suggest that sigma ligands may be utilized to localize and inhibit the proliferation of SCLC tumors.     

Camptothecin-somatostatin conjugates inhibit the growth of small cell lung cancer cells

The effects of camptothecin-somatostatin (CPT-SS) conjugates were investigated on small cell lung cancer (SCLC) cells. CPT was coupled to a SS agonist (SSA), c(Cys-Phe-DTrp-Lys-Thr-Cys)Thr-NH2 using the built in nucleophile assisted-releasing group (L1) N-methyl-aminoethyl-Gly-Dser-Nle-Dtyr-Dser or (L2) aminoethyl-Gly-Dser-Nle-Dtyr-Dser. The resulting CPT-L1-SSA and CPT-L2-SSA inhibited the specific binding of [125I-Tyr11]SS to NCI-H69 cell membranes with IC50 values of 0.2 and 2.1 nM, respectively. [125I]CPT-L1-SSA was internalized by SCLC cells at 37 degrees C but not at 4 degrees C. CPT-L1-SSA and CPT-L2-SSA inhibited in a dose-dependent manner the increase in adenylylcyclase activity caused by 25 microM forskolin. In vitro, 0.3 microM CPT-L1-SSA half-maximally inhibited the clonal growth of SCLC cells and 1 microM CPT-L1-SSA strongly inhibited 3H-thymidine incorporation into DNA and trypan-blue exclusion. These results suggest that CPT conjugated peptides such as CPT-L1-SSA may prove useful for exploring the efficacy of receptor-directed cytotoxicity to inhibit the proliferation of SCLC cells.     

Fatty-monastrol derivatives and its cytotoxic effect against melanoma cell growth

Melanoma is the most dangerous type of skin cancer due to the occurrence of metastases. This work is aimed at studying the effects of the insertion of palmitic and oleic acid chain into monastrol in the melanoma cell line, B16F10. Cells were treated with monastrol, palmitic-monastrol or oleic-monastrol for periods of 0, 24, 48 and 72 h, and the cytotoxic effect was observed for palmitic-monastrol and oleic-monastrol after 24 h. For monastrol the effects were observed in 48 h on B16F10 cells, and in 24 h for a non-tumour cell line, melan-a. In this cell line, fatty-monastrol derivatives were cytotoxic after 24 h of exposure in the same concentrations as B16F10. However, oleic-monastrol inhibited cell growth at 20 µM only after 72 h, in contrast to the B16F10 cell line, in which oleic-monastrol inhibited cell growth at 48 h, showing that at least in this structural modification, melan-a was less sensitive than B16F10. The ability of compounds to induce apoptosis and/or necrosis was measured, and it was observed that monastrol induces apoptosis within 24 h. However, the cells treated with fatty-monastrol derivatives did not remain adhered on the well plate after 3 h of treatment. At this time point, these cells still emitted fluorescence indicating viable cells, suggesting a possible effect of palmitic- and oleic-monastrol in the adhesion proteins found on the cell membrane.