Mice primed by feeding griseofulvin or diethyl 1,4-dihydro 1,4,6-trimethyl 3,5-pyridine decarboxylate for 5 months followed by drug withdrawal for 1 month (drug-primed mice) were given thioacetamide ...intraperitoneally, and the livers were subsequently studied at intervals up to 7 days. The hepatocellular proliferative response was measured by immunostaining for proliferative cell nuclear antigen. Necrosis was followed by measuring ALT. Mallory bodies were identified by immunoperoxidase stains for ubiquitin and cytokeratin. Preneoplastic foci were localized using immunofluorescence stain for glutathione
S-transferase (GST mu) and histochemical stain for gamma glutamyl transpeptidase (GGT). The results showed that the preneoplastic foci selectively proliferated and expanded and formed nodules as indicated by quantitation of nuclei stained positive for proliferating cell nuclear antigen after thioacetamide treatment. Data support the hypothesis that the preneoplastic foci consisted of clones of hepatocytes which preferentially express GST mu, GGT and Mallory bodies. These preneoplastic cells selectively proliferate in response to the promoter effects of necrosis-induced liver cell regeneration (“chemical partial hepatectomy”).
Colorectal cancer is the second most deadly cancer in the United States. When diagnosed early, current treatments bring a limited success; however, once metastasis occurs, radiation and chemotherapy ...are generally ineffective. Structural changes in the ECM are necessary for cell migration during tissue remodeling. Matrix metalloproteinases (MMPs), VEGF, Ki-67 (proliferative protein), and constituents of ECM, such as fibronectin, play a critical role in angiogenesis and are thus crucial in neoplastic invasion and metastasis. Based on antitumor properties of certain nutrients, we investigated the effect of a diet containing lysine, proline, arginine, ascorbic acid, and green tea extract (NM) on the growth of tumors, induced by implanting human colon HCT 116 cancer cells in athymic nude mice, and the expression of MMPs, VEGF, Ki-67 and fibronectin in these tumors, as well as the production of mucin (by PAS staining). After one week of isolation, 5 to 6 week-old athymic male nude mice (n=12) were inoculated with 3x10(6) colon cancer HCT 116 cells. After injection, the mice were randomly divided into 2 groups; group A was fed a regular diet and group B was fed a regular diet supplemented with 0.5% NM. The mice were sacrificed 4 weeks later, and their tumors were excised, weighed, and processed for histology. Results showed that the nutrient mixture (NM) inhibited growth and reduced the size of tumors in nude mice. Furthermore, histological evaluation revealed increased mitotic index, MMP-9 and VEGF secretion and reduced basement membrane in the control group tissues. Nutrient supplementation strongly suppressed the growth of tumors without any adverse effects in nude mice, suggesting the nutrient combination has potential as an anticancer agent. Histological studies supported these findings by showing inhibition of MMP-9 and VEGF secretion and mitotic index, which are critical parameters for cancer control and prevention.
Aims: Bladder cancer, the fourth highest incident cancer in men and tenth in women, is associated with a high rate of recurrence, even when treated in situ, and prognosis is poor once the cancer ...metastasizes to distant sites. Based on anticancer properties, we investigated the effect of a mixture of lysine, proline, arginine, ascorbic acid, and green tea extract on human bladder cancer cells T‐24 by measuring: proliferation, matrix metalloproteinase (MMP) expression, and cancer cell invasive potential.
Methods: Human bladder cancer cells T‐24 (ATCC) were grown in McCoy medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 mg/mL) in 24‐well tissue culture plates. At near confluence, the cells were treated with the nutrient mixture dissolved in media and tested at 0, 10, 50, 100, 500, and 1000 µg/mL in triplicate at each dose. Cells were also treated with PMA 200 ng/mL to study enhanced MMP‐9 activity. Cell proliferation was evaluated by MTT assay, MMP activity by gelatinase zymography, and invasion through Matrigel.
Results: Nutrient mixture inhibited the T‐24 cell secretion of MMP‐2 and ‐9, with virtual total inhibition of MMP‐2 at 500 µg/mL and MMP‐9 at 100 µg/mL. The nutrient mixture significantly reduced the invasion of human bladder cancer cells T‐24 through Matrigel in a dose‐dependent fashion, with 95% inhibition at 500 µg/mL and 100% at 1000 µg/mL nutrient mixture (P < 0.001).
Conclusion: Our results suggest that our nutrient mixture is an excellent candidate for therapeutic use in the treatment of bladder cancer, by inhibiting critical steps in cancer development and spread, such as MMP secretion and invasion.
Current treatments are generally ineffective once breast cancer has metastasized; median survival is reduced to 2-3 yr. Previous research studies demonstrating potent synergistic antitumor activity ...of lysine, proline, ascorbic acid, and epigallocatechin gallate prompted us to investigate the in vivo inhibitory effect of a nutrient mixture containing lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate (NM) on the growth of human cancer xenografts in female athymic nude mice. Five to six week old female mice were inoculated with 3x106 breast cancer cells MDA-MB-231. After injection, the mice were randomly divided into two groups A and B; group A was fed a regular diet and group B with the regular diet supplemented with 0.5% of the nutrient mixture (NM). Four weeks later, the mice were sacrificed, and their tumors were excised, weighed, and processed for histology. We also tested the effect of NM in vitro on estrogen-receptor positive (ER+) MCF-7 and estrogen-receptor negative (ER-) MDA-MB-231 breast cancer cell lines by measuring: cell proliferation by MTT assay, expression of MMPs by gelatinase zymography, invasion through Matrigel, and VEGF by ELISA. MCF-7 cells were also treated with estradiol to study enhanced invasion and expression of MMPs and VEGF. Results showed that NM inhibited the growth and reduced the size of tumors in female nude mice by 27%. Furthermore, histological evaluation revealed increased mitotic index, MMP-9 and VEGF secretion, and PAS material (mucin) in the control group tissues. In vitro studies showed NM inhibited MDA-MB-231 cell growth by 34% at 500 microg/mL and MCF-7 cell growth by 18% at 1000 microg/mL. Invasion of MDA-MB-231 through Matrigel was inhibited by 50%, 60%, and 95% by 10, 50, and 100 microg/mL of NM, respectively. The results of this study demonstrated that the nutrient mixture tested significantly suppressed tumor growth of breast cancer cells in female athymic nude mice and significantly inhibited MMP expression, angiogenesis, and invasion in breast cancer cells, in vitro, offering promise for therapeutic use in the treatment of breast cancer.
Extracellular matrix (ECM) function and structure are severely compromised at atherosclerotic lesion sites, contributing to initiation and progression of the disease. This study investigated whether ...ECM biological properties would be beneficially affected by exposure to nutrients essential for collagen synthesis and posttranslational modification. Confluent layers of human aortic smooth muscle cells (SMC) grown on collagen substrate were cultured in the presence of the tested compounds for 7 to 10 days. Pretreated cells were removed from the ECM surface by differential treatment and replaced with secondary innocent SMC cultures. Secondary SMC growth rate and invasiveness were assayed in standard growth medium. ECM protein composition was assayed immunochemically. ECM produced in the presence of ascorbic acid reduced SMC proliferation in a dose-dependent manner. Plant-derived phenolic extracts expressed different degrees of SMC growth inhibition when present during ECM production. A combination of selected nutrients had a greater effect than did individual components. The ECM deposited by SMC in the presence of ascorbate, lysine, proline, and green tea catechins inhibited SMC migration rate up to 70%. The ECM produced under conditions of chronic essential nutrient deficiency can support proatherosclerotic SMC behavior. A combination of selected nutrients can counteract these adverse effects stronger than individual components.
Current treatment of fibrosarcoma, an aggressive cancer of the connective tissue, is generally associated with poor prognosis. Matrix metalloproteinases (MMPs), vascular endothelial growth factor ...(VEGF), and constituents of the extracellular matrix (ECM), such as fibronectin, play a critical role in angiogenesis and underlie neoplastic invasion and metastasis. This and anticancer properties of lysine, proline, arginine, ascorbic acid, and green tea extract (NM) prompted us to investigate the effect of these nutrients in vitro on human fibrosarcoma cells HT-1080 by measuring cell proliferation, modulation of MMP-2 and MMP-9, and invasive potential. In vivo, we studied the growth of human fibrosarcoma HT-1080 cells in athymic nude mice and the expression of MMPs and VEGF. Cell proliferation was evaluated by MTT assay, MMP expression by gelatinase zymography, and invasion through Matrigel and migration by scratch assay. Tumors were excised, weighed, and processed for histology in both the control and nutrient-supplemented groups. Results showed NM inhibited the growth and reduced the size of tumors in nude mice; decreased MMP-9 and VEGF secretion was found in the supplemented group tissues. NM inhibited invasion through Matrigel and migration with total inhibition at 1,000 microg/mL. These results offer promise in the therapeutic use of the nutrient mixture of lysine, proline, arginine, ascorbic acid, and green tea extract tested in the treatment of fibrosarcoma.
Mammary tumors were developed by intraperitoneal injection of N-methyl-N-nitrosourea (MNU) in 21-day-old, sexually immature female Wistar rats. Injection of MNU was repeated 14 weeks after the first ...one. When palpable tumors were evident in all of the rats, various dietary treatments were initiated for a period of 8 weeks. The treatments were designed to provide 30 mg green tea extract either alone or as a nutrient mixture (E). E was then expanded to include either a nutrient supplement (N), quercetin (Q) or both (N+Q). At the end of the treatment, tumor size/rat measured in the live rats was significantly lower in the groups receiving E, E+Q, E+N and E+N+Q than in the positive control (PC) group which did not receive any dietary treatment. Tumor number/rat, tumor volume/rat and tumor weight/rat were evaluated after sacrificing the rats on the 60th day. The rats receiving E+N+Q showed significantly lower values for the three parameters as compared to the PC group. The PC group showed 24 carcinomas mostly of grade III severity, while the E+N+Q group had only 6 carcinomas, all of which were of grade II severity.
A hallmark of renal cell carcinoma (RCC) invasion is its ability to degrade ECM by local production of gelatinase enzymes. Although many studies on RCC have demonstrated the importance of MMPs, very ...little information is currently known regarding the effect of inducers and inhibitors. We therefore investigated the effect of inducers and inhibitors on RCC 786-0 in vitro. Human RCC 786-0 (ATCC) was grown in RPMI medium supplemented with 10% FBS, penicillin, and streptomycin in 24-well tissue plates. At near confluence, the cells were washed with PBS; the serum-free medium was incubated with various inducers: phorbol ester (PMA), tumor necrosis factor alpha (TNF-alpha), interleukin 1-beta (IL-1beta) and lipopolysaccharides (LPS). Cells were also incubated with inhibitors: EGCG, doxycycline, and a nutrient mixture with and without PMA; retinoic acid, dexamethasone, H-7; actinomycin D, or cyclohexamide. After 24 h, the medium was removed and analyzed for MMP-2 and MMP-9 by gelatinase zymography. RCC 786-0 secreted two bands, a major band corresponding to MMP-2 and a faint band corresponding to MMP-9. PMA and TNF-alpha, with increased concentration, increased MMP-9 secretion, while IL-1beta and LPS did not significantly modify MMP-9 activity. MMP-2 secretion was not affected by any of the inducers. All the inhibitors tested without and with PMA showed a dose-dependent decrease in both MMP-2 and -9 expression. Further studies are in progress to confirm the role of MMP-9 on Matrigel invasion using PMA, cytokines, and LPS.
Abstract
Several previous studies have indicated that the stromal tissue in tumors plays an indispensable role in promoting tumor growth and invasion. This is due to a reciprocal interaction and ...communication between the stromal and neoplastic components of tissues. Even in breast cancer cell invasion and metastases a key event has been shown to be the stimulation of fibroblasts by cancer cells to secrete enzymes such as MMPs to help degrade and modify the extracellular matrix.
In this study, we investigate the in vitro and in vivo effects of two micronutrient combinations - NM1 and NM2 on the inhibition of growth and invasion of human breast cancer cell line MDA-MB-231 alone or in combination with human dermal fibroblasts (NHDF). NM1 contains ascorbic acid, lysine, proline, green tea extract and quercetin while NM2 is a combination of plant extracts such as curcumin, cruciferex, green tea extract and resveratrol.
In the in vitro studies we evaluated the effect of NM1 and NM2 on several parameters such as breast cancer cell proliferation, migration and expression of matrix degrading proteases (MMPs). Treatment of NHDF with conditioned media (CM) from MDA-MB-231 cells resulted in an enhanced MMP2 enzyme expression. However, treatment with both combinations of nutrients resulted in a dose dependent reduction in the MMP2 expression to basal or lower levels. The invasion assay also showed that the invasive ability of normal fibroblasts was significantly enhanced by co-incubation with breast cancer cells in the Boyden chamber. The invasiveness was however reduced considerably upon treatment with both NM1 and NM2. A similar dose dependent inhibition in growth, invasion and migration was observed in MDA-MB-231 alone and in co-cultures.
In the in vivo study, 5-6 week old mice were orthotopically inoculated with 4×106 MDA-MB-231 cells alone (Group 1) or in combination with fibroblasts (Group 2) into the mammary pad of female nude mice. Each group (n = 18) was further divided into 3 sets based on the diet: Set A was fed a regular diet; Set B was fed a diet supplemented with NM1 and Set C was fed a diet supplemented with NM2. After 6 weeks the mice were sacrificed and the tumors were excised and processed for histology. The tumor weight was recorded and it was found that NM1 and NM2 inhibited tumor weight by 42% each in group 1 and by 41% and 55% respectively in group 2. The tumor weight decreased from 1.78±0.28g in group 1A to 1.03±0.25g in group 1B and 1.03±0.12g in group 1C. Similarly, tumor weight showed a reduction from 1.64±0.19g in group 2A to 0.97±0.17g in group 2B and 0.74±0.26g in group 2C.
The results suggest that both NM1 and NM2 have significant therapeutic potential in treatment of breast cancer, both through a direct suppressive effect on cancer cells and through an indirect effect of limiting cancer-stromal interactions by inhibiting communication between cancer cells and fibroblasts.
Citation Format: Neha Shanker, M. Waheed Roomi, Aleksandra Niedzwiecki, Matthias Rath. Inhibition of breast cancer - fibroblast interaction by specific micronutrient combinations. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3194. doi:10.1158/1538-7445.AM2015-3194
Abstract
Lipoprotein(a), composed of LDL and an adhesive protein apo(a), is produced in humans and primates, the species which lost an ability to synthesize vitamin C endogenously. We have shown ...earlier that Lp(a), due to its strong ECM binding properties, may be considered a biological ‘stability’ molecule for the structurally weakened connective tissue in the vascular wall. The development and progression of cancer is characterized by loss of ECM integrity which facilitates tumor growth and metastasis. We developed a unique mouse model lacking endogenous vitamin C production (Gulo-/-) and synthesizing human Lp(a) (lp(a)+), which has been used in this study to investigate the role of Lp(a) and other lipoproteins in cancer.
The female Gulo-/-:Lp(a)+ and control wild type Balb/c mice were orthotopically inoculated with 4T1 breast cancer cells (500,000) The transgenic and control mice were divided into 4 different dietary groups in respect to dietary vitamin C intake: A) low ascorbate intake for 6 weeks; B) high ascorbate intake for 6 weeks; C) low ascorbate intake for 3 weeks followed by high ascorbate for 3 weeks; D)high ascorbate intake for 3 weeks followed by low ascorbate for 3 weeks. Control groups of Lp(a)+;Gulo(-/-) mice without tumor inoculation were put on the same Vitamin C regimens. Wild type controls included mice without and with 4T1 inoculation kept on regular mouse chow for 6 weeks.
After 6 weeks all (100%) wild type mice developed tumors, while 50% of Lp(a)+;Gulo(-/-) mice kept on high ascorbate diet for 6 weeks did not develop primary tumors and in only a small number of mice some residual tumor cells or inflammatory infiltrates were detected in the lungs by histology. In transgenic mice supplemented with low vitamin C for 6 weeks, the reduction of primary tumors incidence was 33% compared to wild type mice. In addition, primary tumors from wild type mice were on average over 2-fold larger (1.80+/-0.62g) than tumors from Lp(a)+;Gulo(-/-) mice on continual low Vitamin C (0.77+/-0.98g) or continual high Vitamin C (0.63+/-1.09g. Primary tumors from Lp(a)+;Gulo(-/-) mice immunostained positively for Lp(a) and their size was inversely proportional Lp(a) cholesterol serum levels. Lp(a) could not be detected in tumors from wild type mice and the presence of tumors was associated with higher LDL serum levels. The results implicate that Lp(a) may play a role in controlling tumor growth and expansion through its ECM adhesive properties.
Citation Format: John Cha, M. Waheed Roomi, Aleksandra Niedzwiecki, Matthias Rath. Lipoprotein(a) and vitamin C affect the development of breast cancer tumors in Lp(a)+; Gulo-/- mice. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2288. doi:10.1158/1538-7445.AM2015-2288
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