Anticancer Activity of Camel Milk via Induction of Autophagic Death in Human Colorectal and Breast Cancer Cells

Abstract

Background/ Objective: Camel milk is traditionally known for its human health benefits and believed to be a remedy for various human ailments including cancer. The study was aimed to evaluate the inhibitory effects of commercially available camel milk on cancer cells and its underlying mechanism(s). Materials and Methods: Two cell lines: colorectal cancer HCT 116 and breast cancer MCF-7 were cultured with different doses of camel milk. The effects of camel milk on cell death were determined by MTT assay, viability by trypan blue exclusion assay and migration by in vitro scratch assay. The mechanism was elucidated by western blotting and confocal microscopy was used to confirm autophagy. Results: Camel milk significantly reduced proliferation, viability as well as migration of both the cells. The accumulation of LC3-II protein along with reduction in expression of p62 and Atg 5-12, the autophagy proteins implied induction of autophagy. The (GFP)-LC3 puncta detected by confocal microscopy confirmed the autophagosome formation in response to camel milk treatment. Conclusion: Camel milk exerted antiproliferative effects on human colorectal HCT 116 and breast MCF-7 cancer cells by inducing autophagy.

Keywords: Colorectal cancer; breast cancer; camel milk; autophagy.

Figure 1

Effect of Camel Milk and Bovine Milk on Cell Viability. (a, b) HCT 116 and MCF-7 cells were seeded and after 2h incubated with varying concentration of camel milk and the number of viable cells were counted after 24, 48 and 72h using trypan blue exclusion assay. Values represent percentage of the control (0 µL/mL) and are shown as mean ± SEM (n=3), *p <0.03, **p <0.003. (c, d) HCT 116 and MCF-7 cells were seeded and after 24h were incubated with 100 and 250 µL/mL of bovine milk and the viable cell count was made after 48h using trypan blue.

Figure 2

Effect of camel milk on cell proliferation. (a, b) HCT 116 and MCF-7 cells were seeded and incubated with various concentration of camel milk for 24, 48 and 72h, thereafter cell proliferation was assessed using MTT assay. Values are presented as percentage of the control (0 µL/mL) and are shown as mean ± SEM (n=3), *p <0.03, **p <0.01, ***p <0.001.

Figure 3

Effect of Camel Milk on Cell Migration, in vitro Scratch Wound Healing Assay. (a, c) HCT 116 and MCF-7 cells were grown in DMEM media to confluence, wounded (t=0h) by using a sterile pipette tip and then treated with various concentration of camel milk. After 21h, the migration of cells into the wound surface were captured under the microscope (magnification, 40x). Scale bar: 200 µm. (b, d): Percentage of wound healing relative to the distance measured in (a) and (c) quantified using Image J. Values are represented as mean ± SEM, **p <0.02, ***p <0.001. Data are representative of triplicate experiments.

Figure 4

Effect of camel milk on apoptotic and autophagy markers. (a, e): HCT 116 and MCF-7 cells were treated with 100 and 250 µL/mL of camel milk for 48h. The expression of PARP and Bcl-2 proteins in the cell lysates were analysed by western blotting. HCT 116: (b-d) and MCF-7: (f-h); the protein lysates were tested for the expression of LC3, p62 and Atg5-12 proteins by western blotting analysis. GAPDH served as the loading control. The band intensities were quantified using Image J and normalised against GAPDH. The data are from triplicate experiments and presented as mean ± SEM, *p <0.03, **p <0.01.

Figure 5

Camel Milk Induces Autophagy. (a, b): Light microscopy images showing comparative morphological changes in HCT 116 and MCF-7 cells untreated or treated with camel milk for 24h. The black arrows indicate the presence of vesicles. Scale bar: 15 µm. (c, d): Confocal microscopy images showing the localization of GFP-LC3 puncta (white arrows) in HCT 116 and MCF-7 cells treated with camel milk in compared to the diffused fluorescence in the untreated cells. Cells were transiently transfected with pEGFP-LC3 plasmid for 24h and then the GFP-LC3 positive cells were treated with or without 100 µL/mL camel milk for additional 24h. Fluorescence images were captured at 60x magnification, scale bar: 20 µm.

Figure 6

Schematic Representation of Autophagic Flux and Formation of Autophagosomes Emphasising the Role of Various Proteins Involved. Microtubule-associated protein 1 light chain 3 (LC3) precursors are processed to form LC3-I is lipidated by phosphatidyl ethanolamine (PE) forms the active LC3-II which is then localized onto the double membrane vesicles that forms the nascent autophagosomes. The autophagy proteins such as Atg5 and Atg12 forms a complex (Atg5-12), then gets directed onto to the double membrane vesicles, further mediates elongation process leading to the autophagosome formation. The protein p62 (sequestosome 1) co-localizes with the ubiquitinated proteins (fated to be degraded) gets sequestered into the double membrane vesicles, subsequently engulfs into the autophagosomes destined for degradation.