The Antimetastatic and Antiangiogenesis Effects of Kefir Water on Murine Breast Cancer Cells

Abstract

Background: Kefir is a unique cultured product that contains beneficial probiotics. Kefir culture from other parts of the world exhibits numerous beneficial qualities such as anti-inflammatory, immunomodulation, and anticancer effects. Nevertheless, kefir cultures from different parts of the world exert different effects because of variation in culture conditions and media. Breast cancer is the leading cancer in women, and metastasis is the major cause of death associated with breast cancer. The antimetastatic and antiangiogenic effects of kefir water made from kefir grains cultured in Malaysia were studied in 4T1 breast cancer cells.

Methods: 4T1 cancer cells were treated with kefir water in vitro to assess its antimigration and anti-invasion effects. BALB/c mice were injected with 4T1 cancer cells and treated orally with kefir water for 28 days.

Results: Kefir water was cytotoxic toward 4T1 cells at IC₅₀ (half-maximal inhibitory concentration) of 12.5 and 8.33 mg/mL for 48 and 72 hours, respectively. A significant reduction in tumor size and weight (0.9132 ± 0.219 g) and a substantial increase in helper T cells (5-fold) and cytotoxic T cells (7-fold) were observed in the kefir water-treated group. Proinflammatory and proangiogenic markers were significantly reduced in the kefir water-treated group.

Conclusions: Kefir water inhibited tumor proliferation in vitro and in vivo mainly through cancer cell apoptosis, immunomodulation by stimulating T helper cells and cytotoxic T cells, and anti-inflammatory, antimetastatic, and antiangiogenesis effects. This study brought out the potential of the probiotic beverage kefir water in cancer treatment.

Keywords: 4T1 cell; antiangiogenesis; antimetastasis; breast cancer; immunomodulatory; kefir; kefir water.

Figure 1.

Tumor size, weight (g) and volume (cm³), obtained from 4T1 cell-challenged mice. (A) Images of tumor harvested from untreated group and kefir water–treated group after 28 days. (B) Tumor weight of untreated group and kefir water–treated group after 28 days. (C) Tumor volume of untreated group and kefir water–treated group after 28 days. Each value represents the mean ± SD (n = 7 mice per group). Significant difference (*P < .05) from the untreated group was calculated using the t-test.

Figure 2.

Hematoxylin and eosin stain image of tumor from untreated group and kefir water–treated group. Black circles show mitotic cells. (A) The histogram shows the number of mitotic cells per tumor section in the untreated group and kefir water–treated group (n = 3 slides per mice). Significant difference (*P < .05) from the untreated group was calculated using the t-test. (B) Terminal deoxynucleotidyl transferased UTP nick-end labeling (TUNEL) assay image of the tumor from untreated and kefir water–treated groups. Black circles show DNA fragmentation and apoptotic cells (dark brown spots). The histogram shows the number of apoptotic cells per tumor section in the untreated group and kefir water–treated group (n = 3 slides per mice). Significant difference (*P < .05) from the untreated group was calculated using the t-test.

Figure 3.

(A) The images of clonogenic colonies in lung harvested from 4T1 cell–challenged mice at a dilution factor of 10⁴ and the number of 4T1 colonies formed after 10 days of incubation (n = 6 mice per group). Significant difference (*P < .05) from the untreated group was determined using the t-test. (B) Bone marrow smear for untreated and kefir water–treated group at 200× and 400× magnification.

Figure 4.

The results from cytokine (IL-10, IFN-γ, IL-1β, IL-2) enzyme-linked immunosorbent assay (ELISA) assay on blood serum from the untreated cancer group and kefir water–treated cancer group. Data are presented as means ± SD (n = 6 mice per group). Significant difference (*P < .05) from the untreated group was determined using the t-test.

Figure 5.

Flow cytometry dot plots and histograms of immune markers (CD3, CD4, and CD8) on splenocytes harvested from the normal group, untreated cancer group, and kefir water–treated cancer group. Data are presented as means ± SD. Significant difference (*P < .05) from the untreated group was determined using the t-test.

Figure 6.

(A) NO levels in tumors from the untreated and kefir water–treated groups. (B) Malonaldehyde (MDA) levels in tumors from the untreated group and kefir water–treated group. Data are presented as means ± SD. Significant difference (*P < .05) from the untreated group was determined using the t-test.

Figure 7.

(A) Relative mRNA expression levels of ICAM, iNOS, MMP-9, IL-1β, NF-κB, G-CSF, GM-CSF, IL-4, and TNF-α. Each value represents the mean ± SD. Significant difference (*P < .05) from the untreated group was determined using the t-test. The experiment was done in triplicate. (B) The angiogenesis-related proteome array test was performed to evaluate the level of change of selected proteins. Data are presented as means ± SD. Significant difference (*P < .05) from the untreated group was determined using the t-test.