John Cha

Ascorbate supplementation inhibits growth and metastasis of B16FO melanoma and 4T1 breast cancer cells in vitamin C-deficient mice

Degradation of the extracellular matrix (ECM) plays a critical role in the formation of tumors and metastasis and has been found to correlate with the aggressiveness of tumor growth and invasiveness of cancer. Ascorbic acid, which is known to be essential for the structural integrity of the intercellular matrix, is not produced by humans and must be obtained from the diet. Cancer patients have been shown to have very low reserves of ascorbic acid. Our main objective was to determine the effect of ascorbate supplementation on metastasis, tumor growth and tumor immunohistochemistry in mice unable to synthesize ascorbic acid [gulonolactone oxidase (gulo) knockout (KO)] when challenged with B16FO melanoma or 4T1 breast cancer cells. Gulo KO female mice 36-38 weeks of age were deprived of or maintained on ascorbate in food and water for 4 weeks prior to and 2 weeks post intraperitoneal (IP) injection of 5×105 B16FO murine melanoma cells or to injection of 5×105 4T1 breast cancer cells into the mammary pad of mice. Ascorbate-supplemented gulo KO mice injected with B16FO melanoma cells demonstrated significant reduction (by 71%, p=0.005) in tumor metastasis compared to gulo KO mice on the control diet. The mean tumor weight in ascorbate supplemented mice injected with 4T1 cells was reduced by 28% compared to tumor weight in scorbutic mice. Scorbutic tumors demonstrated large dark cores, associated with increased necrotic areas and breaches to the tumor surface, apoptosis and matrix metalloproteinase-9 (MMP-9), and weak, disorganized or missing collagen I tumor capsule. In contrast, the ascorbate-supplemented group tumors had smaller fainter colored cores and confined areas of necrosis/apoptosis with no breaches from the core to the outside of the tumor and a robust collagen I tumor capsule. In both studies, ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum inflammatory cytokine interleukin (IL)-6 (99% decrease, p=0.01 in the B16F0 study and 85% decrease, p=0.08 in the 4T1 study) compared to the levels in gulo KO mice deprived of ascorbate. In the B16FO study, ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum VEGF (98% decrease, p=0.019 than in the scorbutic gulo KO mice). As expected, mean serum ascorbate level in ascorbate-restricted mice was 2% (p<0.001) of the mean ascorbate levels in supplemented mice. In conclusion, ascorbate supplementation hinders metastasis, tumor growth and inflammatory cytokine secretion as well as enhanced encapsulation of tumors elicited by melanoma and breast cancer cell challenge in gulo KO mice.

Effects of a nutrient mixture on immunohistochemical localization of cancer markers in human cervical cancer HeLa cell tumor xenografts in female nude mice

Although fully treatable in the early stages, once cervical cancer has metastasized, patient outcome is poor. The main objective of this study was to examine the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on HeLa cell xenografts in nude female mice. Tumor growth was measured and immunohistochemical staining was evaluated for the following cancer markers: Ki67 (proliferation); matrix metalloproteinase (MMP)-2 and -9 (invasion/metastasis); vascular endothelial growth factor (VEGF) (angiogenesis); terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and B-cell lymphoma 2 (Bcl-2) (apoptosis); cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) (inflammation); and glutathione S-transferase π (GSTπ) (a general cancer marker). Following housing for a week, 5/6-week-old female athymic nude mice (n=12) were inoculated subcutaneously with 3×106 HeLa cells in 0.2 ml phosphate-buffered saline and 0.1 ml Matrigel™ and randomly divided into two groups; control group mice were fed regular mouse chow and NM group mice the regular diet supplemented with 0.5% NM (w/w). After four weeks, the mice were sacrificed and their tumors were excised and processed for histology. The NM strongly inhibited the growth of HeLa xenografts in nude mice. The mean tumor weight was reduced to 59% (P=0.001) in the mice fed the NM compared with the tumor weight in the controlled diet mice. Ki67, MMP-2 and -9, VEGF, TUNEL, Bcl-2, COX-2, iNOS and GSTπ all showed a lower intensity and frequency of staining in the NM group compared with that in the control group. In conclusion, NM supplementation strongly inhibited tumor growth and cancer markers in female nude mice injected with HeLa xenografts.

In vitro and in vivo effects of a nutrient mixture on breast cancer progression

Long-term survival of patients with breast cancer remains poor, due to metastasis and recurrence. We investigated the effects of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract in vitro and in vivo on 4T1 murine breast cancer, a representative model for metastatic breast cancer. After one week of isolation, 5-6-week-old female Balb/C mice were inoculated with 5x10⁵ 4T1 cells into the mammary pad and randomly divided into two groups; the control group was fed a regular diet and the NM group a regular diet supplemented with 0.5% NM. After four weeks, the mice were sacrificed and their tumors, lungs, livers, kidneys, hearts and spleens were excised and processed for histology. Dimensions (length and width) of tumors were measured using a digital caliper, and the tumor burden was calculated using the following formula: 0.5 x length x width. We also tested the effect of NM in vitro on 4T1 cells, measuring cell proliferation by MTT assay, MMP secretion by zymography, invasion through Matrigel, migration by scratch test and morphology by H&E staining. NM inhibited tumor weight and burden of 4T1 tumors by 50% (p=0.02) and 53.4% (p≤0.0001), respectively. Lung metastasis was profoundly inhibited by NM supplementation: mean number of colonies was reduced by 87% (p<0.0001) and mean weight of lungs by 60% (p=0.0001) compared to control mice. Metastasis to liver, spleen, kidney and heart was significantly reduced with NM supplementation. In vitro, NM exhibited 50% toxicity over the control at 250 and 500 µg/ml concentrations. Zymography demonstrated MMP-2 and MMP-9 secretion which was inhibited by NM in a dose-dependent manner, with virtual total inhibition of both at 1,000 µg/ml. Migration by scratch test and invasion through Matrigel were inhibited in a dose-dependent manner with total block of invasion at 250 and of migration at 1,000 µg/ml. These results suggest that NM has therapeutic potential in the treatment of breast cancer.

Evolution of angiotensin II-mediated atherosclerosis in ApoE KO mice

The dysregulation of renin-angiotensin system-signaling is a contributing factor to the development of atherosclerosis and cardiovascular disease. We studied the progression of angiotensin II (AngII)-induced arterial wall atherosclerosis after its induction. Atherosclerosis was accelerated in ApoE (-/-) C57B6 mice by 4 weeks of continuous osmotic pump administration of AngII (23 nmol/24 h). Changes in aortae were evaluated in these mice at 4 and 16 weeks after pump installation, as well as in control mice that were not administered AngII. In comparison to the non-AngII controls, AngII supplementation induced additional lesions in the aortic arch and its vessels 4 weeks after pump installation, indicating that AngII had an effect. These observations were grossly visible and were confirmed through histological analysis. Additionally, the AngII-mediated injuries continued to accelerate great vessel atherosclerosis after the discontinuation of AngII administration at 4 weeks. Atherosclerosis in the ApoE KO mice at 16 weeks post-AngII administration was increased by over 3-fold compared to the difference between lesion development in the ApoE KO mice at 4 weeks post-AngII and the age-matched control ApoE KO mice. While lesion size 12 weeks after cessation of AngII (16 week post-AngII group) increased 249% compared to the 4 week post-AngII group, differences between the rate of lesion formation in the 4 week post-AngII and non-AngII groups indicated a 70% increase and a linear relationship with time. Aortic dissections were present at 4 weeks post-AngII pump installation at high frequency compared to the unsupplemented controls, in which they were absent. Unexpectedly, aortic dissections were absent at 16 weeks post-AngII, indicating the presence of a healing process. The study suggests that excessive AngII initiates a cascade of pathological biochemical events and plaque evolution that does not cease even after AngII administration is discontinued.

Lipoprotein(a) and vitamin C impair development of breast cancer tumors in Lp(a)+; Gulo-/- mice

Cancer progression is characterized by loss of extracellular matrix (ECM) integrity, which is a precondition for tumor growth and metastasis. In order to elucidate the precise mechanisms of ECM degradation in cancer we used a genetically modified mouse mimicking two distinct human metabolic features associated with carcinogenesis, the lack of endogenous vitamin C synthesis and the production of human Lp(a). Female Lp(a)+; Gulo(−/−) and control wild-type Balb/c mice without these two metabolic features were orthotopically inoculated with 4T1 breast cancer cells (5×105). The transgenic and control mice were divided into 4 different dietary groups in respect to dietary vitamin C intake: i) low ascorbate intake for 6 weeks; ii) high ascorbate intake for 6 weeks; iii) low ascorbate intake for 3 weeks followed by high ascorbate for 3 weeks; iv) high ascorbate intake for 3 weeks followed by low ascorbate for 3 weeks. After 6 weeks, all wild-type mice developed tumors. In contrast, Lp(a)+; Gulo(−/−) mice developed one third less primary tumors (low ascorbate diet) or no primary tumors at all (high ascorbate diet). Significantly, tumors from Lp(a)+; Gulo(−/−) mice immunostained positively for Lp(a) and their size was inversely proportional to Lp(a) serum levels. The results implicate that Lp(a) may play a role in controlling tumor growth and expansion. The most likely mechanism is the competitive inhibition of plasmin-induced ECM degradation due to the homology of Lp(a) components to plasminogen. The confirmation of this pathomechanism could lead to a universal therapeutic target for the prevention and treatment of cancer.

Abstract 2288: Lipoprotein(a) and vitamin C affect the development of breast cancer tumors in Lp(a)+; Gulo-/- mice

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

Nutrient supplementation modulates angiotensin II-mediated atherosclerosis in ApoE KO mice

We studied the effect of nutrient/herbal extract mixture (NM) supplementation prior to angiotensin II (AngII) administration on ApoE KO mice. AngII-mediated injuries, which included increased atherosclerotic lesion size (by 31%) and severity (by 200%), as well as an increased number of necrotic cores (by 44%), were attenuated by NM supplementation, resulting in the reduction of lesion size by 20% (p=0.09), lesion severity by 31% (p=0.13) and the average number of necrotic cores per arterial tree by 39% (p=0.35), compared to mice fed the regular diet. The frequency of Type B thoracic aortic dissections and abdominal aneurysms in AngII-treated mice was reduced from 55.6% to 14.3 and 42.97%, respectively, with NM supplementation. Macrophage density in the aortic lesions of NM-supplemented AngII-treated mice was 20% lower (p=0.033) than in mice on the regular diet, whereas collagen density in NM mice was increased by 208% (p=0.026) compared to that in mice fed the regular diet, making for a more stable plaque type. NM-supplemented mice showed a significant reduction in total cholesterol (32% decrease, p=0.0001) and LDL (66% decrease, p<0.0001), commonly measured risk factors for cardiovascular disease. In conclusion, NM consumption prior to AngII administration in ApoE KO mice minimized aortic dissections, inflammatory monocyte recruitment to plaques, plaque mass and abdominal aortic dilation.

Abstract 4963: In vivo and in vitro effect of a nutrient mixture on murine 4T1 breast cancer

Breast cancer patients often have detectable or occult metastases at diagnosis and most patients will develop metastatic lesions during the course of the disease. We investigated the effect of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract on murine breast cancer 4T1, a unique metastatic breast cancer model that has the capacity to metastasize efficiently to sites affected in human breast cancer. After one week of isolation, 5-6 week old female Balb/C mice were inoculated with 5x105 4T1 cells into the mammary pad and randomly divided into two groups; group A was fed a regular diet and group B a regular diet supplemented with 0.5% NM. After four weeks, the mice were sacrificed and their tumors, lungs, livers, kidneys, hearts and spleens were excised and processed for histology. Dimensions (length and width) of tumors were measured using a digital caliper, and the tumor burden was calculated using the following formula: 0.5 x length x width. We also tested the effect of NM in vitro on 4T1 cells, measuring cell proliferation by MTT assay, MMP secretion by zymography, invasion through Matrigel , migration by scratch test and morphology by HE 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4963. doi:10.1158/1538-7445.AM2014-4963

Abstract 2822: Ascorbic acid synergistically potentiates antimetastatic effect of natural products on 4T1 tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Our main objective was to determine the differential response of female gulonolactone oxidase (Gulo) mice challenged with 4T1 to vitamin C deficient, subclinical vitamin C sufficient, or vitamin C combined with other additional natural products (EQC) diets against 4T1 secondary growth phase metastasis to lungs, kidneys, and liver. Live murine 4T1 cells (5x105) were administered subcutaneously to the right flank of female Gulo (-/-) mice (n=27) 35-40 weeks of age and 18 age matched female wild-type mice. Tumors were established for an additional 2 weeks on subclinical vitamin C (100mg/L in water) in the Gulo (-/-) group, or with regular food and water in the wild-type vitamin C-generating mice. Tumor measurements were taken and the Gulo (-/-) mice were distributed into 3 groups (n=9 in each) to ensure average tumor burden was equivalent before therapy. Mice were then maintained for an additional 2 weeks on either subclinical vitamin C in water, subclinical C water + vitamin C (equivalent to that provided by 0.5% EQC supplemented diet), and subclinical C water + 0.5% EQC supplemented diet. Wild-type mice received an additional 2 weeks of either regular murine diet or 0.5% EQC supplemented food and regular water. At the endpoint, serum was collected, lungs, livers, and kidneys were evaluated for metastatic burden, and tissues collected for histology. Tumors were also harvested, weighed, and sectioned for histology. EQC exhibited the strongest anti-metastatic effect upon the second growth phase in scorbutic Gulo (-/-) mice as well as wild type vitamin C generating mice. Gulo (-/-) mice maintained throughout the study on subclinical vitamin C presented with the worst outcomes, including novel renal involvement. Vitamin C in diet equivalent to that in 0.5% EQC afforded only protection against novel renal pathology, but the metastatic burden was similar to that in subclinical C group. 0.5% EQC abrogated the cases of moderate to severe metastasis in scorbutic Gulo (-/-) mice by 16% compared to subclinical vitamin C group. In wild-type mice, 0.5% EQC abrogated the cases of moderate to severe metastasis by 37%. This study demonstrates that 0.5% EQC is superior to vitamin C alone in reducing metastasis from a primary 4T1 tumor and that prior scurvy is deleterious upon host resistance to primary tumors and reduces the efficacy of subsequent therapy against metastasis. Prior endogenous vitamin C generation in wild-type mice before tumor inoculation and continuous vitamin C generation during therapy with 0.5% EQC suggests that vitamin C both enhances host resistance and synergistically potentiates the effect of additional therapy. Tumor mass itself was not related to metastatic burden, suggesting that differing biochemical composition of the stromal and tumor structures are responsible for a metastatic or non-metastatic tumor of equal size. Citation Format: John Cha, M. Waheed Roomi, Matthias Rath, Aleksandra Niedzwiecki. Ascorbic acid synergistically potentiates antimetastatic effect of natural products on 4T1 tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2822. doi:10.1158/1538-7445.AM2013-2822