The effect of CELLFOODTM on radiotherapy or combined chemoradiotherapy: preclinical evidence

Abstract

Background: Based on previous observations that the nutraceutical CELLFOOD™ (CF), the 'physiological modulator' that aimed to make oxygen available 'on demand', inhibits the growth of cancer cells, this study was designed to investigate the role of CF in the regulation of hypoxia-inducible factor 1 alpha (HIF1α) and its correlated proteins, phosphoglycerate kinase 1 and vascular endothelial growth factor. Our idea was that CF, acting on HIF1α, in combination with current anticancer therapies could improve their effectiveness.

Methods: To evaluate the effect of CF in association with radiotherapy and chemotherapy, different human cancer cell lines and mice with mesothelioma were analysed by tumour growth, clonogenic assay, western blot and immunohistochemical analysis.

Results: CF in combination with radiation with or without cisplatin increases the death rate of cancer cells. In vivo, 70% of mice treated with CF before the mesothelioma graft did not show any tumour growth, indicating a possible preventive effect of CF. Moreover, in mouse mesothelioma xenografts, CF improves the effect of radiotherapy also in combination with chemotherapy treatment. Immunohistochemical analysis of tumour explants showed that HIF1α expression was reduced by the combination of CF and radiotherapy treatment and even more by the combination of CF and radiotherapy and chemotherapy treatment. Mechanistically, CF increases the fraction of oxygenated cells, making the radiotherapy more effective with a greater production of reactive oxygen species (ROS) that in turn, reduce the HIF1α expression. This effect is amplified by further increase in ROS from chemotherapy.

Conclusions: Collectively, results from preclinical trials suggest that CF could be a useful intervention to improve the efficacy of radiotherapy or combined treatment strategies and could be a promising treatment modality to counteract cancer.

Keywords: CELLFOODTM; HIF1α; combined treatment strategies; mesothelioma; radiosensitivity.

Figure 1.

Schematic representation of CD1 nude mice treatments according two different combinations of CF and RT dose fraction. (a) Schedule to evaluate the effect preventive of CF and identify the better treatment experimental conditions using 8 Gy in RT(8)-treated animal groups and different CF doses; (b) schedule of treatment to evaluated the effectivity of the different therapeutic combination with 4 Gy in RT(4) groups and different CF doses. Red cloud indicates the irradiation; syringe indicates the chemotherapeutic treatment; continuous line indicates the daily treatment with CF. Arrows indicate the days; treatments are indicated in the left of the scheme and the name of each treatment group is reported on the right. MSTO, MSTO-211H mesothelioma cell line; CF, CELLFOOD™; CFID, increasing doses of CF; RT, irradiation treatment; RT(8), RT with 8Gy; RT(4), RT with 4 Gy; ST, standard therapy; CNTR, with vehicle.

Figure 2.

CF modulated HIF1α, PGK1 and VEGF expression in cancer cell lines. Western blot analysis of HIF1α, PGK1 and VEGF in Cal27, MSTO and HCT-116 (HCT) cells. (a) HIF1α, PGK1 and VEGF expression basal and after treatment of Cal27, MSTO and HCT cells with 100 µmol/l CoCl₂ for 4, 6, 24 h; (b) HIF1α modulation under hypoxia (CoCl₂) after 4 h of treatment with CF (5 μl per ml of medium corresponding to a 1:200 dilution); (c) PGK1 and VEGF expressions under hypoxia (CoCl₂) after 24 h of treatment with CF (5 μl per ml of medium corresponding to a 1:200 dilution). Cal27, carcinoma of the tongue cell line; MSTO, MSTO-211H mesothelioma cell line; HCT-116, human colon carcinoma cell line; CoCl2, cobalt dichloride; HIF1α, hypoxia-inducible factor 1 alpha; PGK1, phosphoglycerate kinase 1; VEGF, vascular endothelial growth factor. CF, CELLFOOD™.

Figure 3.

CF in combination with radiation increases the tumour cells death. (a) The histograms for each cell line represent the percentage of live cells after CF [RT(0) + CF] or RT(4) or RT(4) + CF, RT(6) or RT(6) + CF treatments compared to untreated CNTR [RT(0) + 0CF, *p < 0.05 versus CF [(RT(0)+CF] or RT treatment [RT(4), RT(6)] alone; 100% of live cells]; (b) clonogenic assay: 500 viable cells untreated CNTR [RT(0) + 0CF] and pretreated with CF [RT(0) + CF], RT(4) and RT(4) + CF, were allowed to grow in normal medium for 10–14 days and then stained by crystal violet solution. The image is representative of three independent experiments. HCT, HCT-116 human colon carcinoma cell line; MSTO-211H, mesothelioma cell line; Cal27, carcinoma of the tongue cell line; Calu3, lung adenocarcinoma cell line; MDA-361, breast cancer cell line; CF, CELLFOOD™; RT, irradiation treatment; RT(0)+0CF, with vehicle; RT(4), RT with 4 Gy; RT(6), RT with 6 Gy; CNTR, with vehicle.

Figure 4.

Combined CF, CISP and irradiation treatment increases cancer cell death. Histograms of MSTO (a) and Cal27 (b) cells survival percentage after the treatment with cisplatin doses ranging between 0.78 and 12.5 µmol/l and CF at dose fixed alone and in association compared to the untreated (CNTR, cell survival 100%). Data are expressed as mean ± SD (standard deviation) of at least three independent experiments. *p < 0.05 versus CNTR and # versus cisplatin (CISP). Histograms of MSTO (c) and Cal27 (d) cells survival after the treatment with 0.78 µmol/l CISP, CF and RT(4) alone or in association. Data are expressed as mean ± SD of at least three independent experiments. *p < 0.05 versus CF. #p < 0.05 versus CISP. °p < 0.05 versus 4GY. §p < 0.05 versus CISP + 4GY. MSTO, MSTO-211H mesothelioma cell line; Cal27, carcinoma of the tongue cell line; CISP, cisplatin; CF, CELLFOOD™; CNTRL, with vehicle. 4GY, irradiation treatment (RT) with 4GY.

Figure 5.

Effect of ROS on HIF1α, PGK1 and VEGF expression. (a) The histogram shows the results of three independent experiments: the percentage of levels of ROS with respect to untreated MSTO cells (CNTR) after the addition of 50–200 μmol/l H₂O₂ (−NAC) and after cotreatment with 5 mmol/l NAC (+NAC). Data are expressed as mean ± SD of at least three independent experiments. ROS effect on the expression of the HIF1α protein, PGK1 and VEGF was detected by western blot in all experiments, (b) represents a single experiment. The relative bands intensities of the proteins of interest after 50–200 μmol/l H₂O₂ (c) and 50–200 μmol/l H₂O₂ with NAC (d) were quantified by the Scion Image software in all experiment. ^*p < 0.05 versus CNTR. CNTR, with vehicle; HIF1α, hypoxia-inducible factor 1 alpha; NAC, N-acetyl-L-cysteine; PGK1, phosphoglycerate kinase 1; ROS, reactive oxygen species; SD, standard deviation; VEGF, vascular endothelial growth factor.

Figure 6.

Effect of CF alone and in association with RT on mesothelioma xenograft mice. The chart, on the left side (a), shows tumour mass growth after the different treatments with CF as described in methods. On the right side: immunohistochemistry of tumour explants (b), the scale bar is 30 µmol/l: (1) haematoxylin–eosin staining of CNTR and CFCNTR; (2) immunohistochemical staining with CD45 of CNTR; (3) and CFCNTR, (4); (c) the graph shows the tumour growth of mice upon treatment with CFID or RT(8) or CFID + RT(8) and untreated (CNTR); CFID, RT(8) and CFID + RT(8) were significantly effective *in reducing tumour growth versus CNTR. (d) the graph shows the average survival for each mice group according to the treatment. *p < 0.05 versus CNTR CNTR, with vehicle; CFCNTR, CF treatment only before xenograft, CF, CELLFOOD™ treatment; CFCF, CF treatment before and after xenograft, CFID, increasing doses of CF; RT(8), irradiation treatment with 8 Gy.

Figure 7.

Effect of CF in association with therapeutic treatments on tumour growth and HIF1α, PGK1 and VEGF expression in mesothelioma mice xenograft. (a) Action of the CF alone and with RT(4) on the tumour growth. * significant versus CNTR, ^¤significant versus CNTR + RT(4); (b) action of the ST alone and with CF on the tumour growth; ^*significant versus CNTR; (c) action of the RT(4) alone, with ST and with ST + CF on the tumour growth; ^*significant versus CNTR + RT(4); ^¤significant versus ST + RT(4); (d) a representative western blot of HIF1α, PGK1 and VEGF of masses removed by mice untreated and treated with ST, RT(4) and CF alone and in combination, and quantification of protein band intensities by Scion Image software and tubulin-normalization (e); (f) immunohistochemistry of tumour explants; the scale bar is 30 µmol/l. Data are expressed as mean ± SD (standard deviation) of all determinations of explants for each group. ^*p < 0.05 versus RT(4). ^#p < 0.05 versus CF + RT(4). CF, CELLFOOD™; CNTR, with vehicle; RT(4), irradiation treatment (RT) with 4 Gy; RT(8), RT with 8 Gy; ST, standard therapy; HIF1α, hypoxia-inducible factor 1 alpha; PGK1, phosphoglycerate kinase 1; VEGF, vascular endothelial growth factor; H/E, haematoxylin and eosin staining.

Figure 8.

Schematic representation of a model explaining the association between CF and radio- and chemotherapy. (a) Before irradiation (baseline), the tumour mass consists of well-oxygenated (green) and nonoxygenated (red) cells. RT kills a higher number of well-oxygenated than hypoxic cells with ROS production and increased HIF1α expression. In the case of CF + RT treatment, CF is expected to increase the fraction of oxygenated cells reducing the hypoxic fraction, making RT more effective (b). This implies a greater production of ROS and reduction of HIF1α (b) compared with RT alone (a). After the CF administration, when ST is added to RT (c), we expected a further increase in ROS, attributable to the action of CISP and an overall reduction in HIF1α expression. ROS, reactive oxygen species; HIF1α, hypoxia-inducible factor 1 alpha; RT, irradiation therapy; CF, CELLFOOD™; ST, standard therapy.