The anticancer activity of milk rich in conjugated linoleic acid

[A atividade anticâncer do leite rico em ácido linoleico conjugado]

ABSTRACT

Milk samples enriched in conjugated linoleic acid (CLA) were obtained from buffalos fed on special diet supplemented with olive oil and sunflower oil mixture (1:1) with monensin oil to enhance the production of CLA. The effect of CLA rich milk on cancer growth was studied both in vitro and in vivo. For the in vitro study, the viability of 3 cancer cell lines, HeLa, HepG-2, and MCF-7, and the viability was examined by neutral red assay following the culture with special media containing CLA rich milk. For the in vivo study, ninety female mature mice were used in two experiments, 45 mice in each experiment, to investigate the effect of feeding milk rich in CLA on life span and the survival rate of mice transplanted with Ehrlich Ascites Carcinoma cells (EACC). Results of the in vitro study showed that both normal milk as well as milk rich in CLA enhanced cell survival of the cancer cell lines. However, the viability of these cells was significantly reduced (80%) when lipid fractions of milk samples were used instead of whole milk. This effect may be due to the essential fatty acid component of CLA rich milk which enhanced the inhibition of cancer cells’ growth. Our results were confirmed when the viability of cancer cells was significantly decreased when CLA was added to their incubation media. Data from the in vivo study showed that the life span of tumor transplanted mice fed milk rich in CLA was longer than that of mice transplanted fed either a normal diet or fed normal milk diets. We concluded that milk rich in CLA has clear anticancer activity in vitro and in vivo. Our results reveal that the action is due to the unsaturated fatty acids, especially CLA which may enhance cancer cells to enter apoptosis.

Keywords:
CLA; Cancer; EACC; HeLa cells; HepG-2; MCF-7; Anticancer

RESUMO

Amostras de leite enriquecidas em ácido linoleico conjugado (CLA) foram obtidas de búfalas alimentadas com uma dieta especial suplementada com azeite de oliva e mistura de óleo de girassol (1:1) com óleo de monensina para aumentar a produção de CLA. O efeito do leite rico em CLA sobre o crescimento do câncer foi estudado in vitro e in vivo. Para o estudo in vitro, a viabilidade de três linhas de células de câncer, HeLa, HepG-2 e MCF-7, e a viabilidade foi examinada pelo ensaio de vermelho neutro após a cultura com meios especiais contendo leite rico em CLA. Para o estudo in vivo, noventa camundongos fêmeas maduros foram usados em dois experimentos, 45 camundongos em cada experimento, para investigar o efeito da alimentação com leite rico em CLA sobre o tempo de vida e a taxa de sobrevivência de camundongos transplantados com células de carcinoma de ascite de Ehrlich (EACC). Os resultados do estudo in vitro mostraram que tanto o leite normal quanto o leite rico em CLA aumentaram a sobrevivência celular das linhas de células cancerígenas. No entanto, a viabilidade dessas células foi significativamente reduzida (80%) quando as frações lipídicas das amostras de leite foram usadas em vez do leite integral. Esse efeito pode ser devido ao componente de ácido graxo essencial do leite rico em CLA, que aumentou a inibição do crescimento das células cancerígenas. Nossos resultados foram confirmados quando a viabilidade das células cancerosas foi significativamente reduzida quando o CLA foi adicionado ao meio de incubação. Os dados do estudo in vivo mostraram que o tempo de vida dos camundongos transplantados com tumores alimentados com leite rico em CLA foi maior do que o dos camundongos transplantados alimentados com uma dieta normal ou com dietas com leite normal. Concluímos que o leite rico em CLA tem uma clara atividade anticâncer in vitro e in vivo. Nossos resultados revelam que a ação se deve aos ácidos graxos insaturados, especialmente ao CLA, que pode aumentar a apoptose das células cancerígenas.

Palavras-chave:
CLA; câncer; EACC; células HeLa; HepG-2; MCF-7; anticâncer

INTRODUCTION

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid, which is preferentially found in dairy products and meat (foods derived from ruminants). Studies indicate that CLA was a powerful anticarcinogen in the rat mammary tumor model with an effective range of 0.1-1% fat in the diet. This protective effect of CLA was noted even when exposure is limited to the time of weaning to carcinogen administration (Ip et al., 1994a; Souza Godinho et al., 2024). Conjugated linoleic acid (CLA) is unique because it is present in food from animal sources, and its anticancer efficacy is expressed at concentrations close to human consumption levels (Belury 1996). Also, CLA which is composed of positional and stereoisomers of octadecadienoate (18:2); reduced chemically induced tumorigenesis of mammary, skin, and colon when a mixture of isomers is fed to these experimental animals (Ip et al., 1996). Many isomers of CLA are readily metabolized to desaturated/elongated products as well as β-oxidized products, suggesting that these metabolites may be important anticancer compounds (Belury 2002).

Results of Larson et al. (2005) showed that high intakes of high-fat dairy foods and CLA may reduce the risk of cancer. So far it has been assumed that CLA is involved in the three stages of carcinogenesis (initiation, promotion, progression) (Pophaly et al., 2022), and the effect differs according to CLA isomers, type and site of the cell organ, and stage of carcinogenesis (Lee and Lee 2005). The anti-tumor potential of docetaxol by conjugated linoleic acids (CLAs) in breast cancer cells in vitro was observed by Fite et al. (2007). Their results showed that CLA isomers augment anti-tumor effects similar to docetaxol in breast cancer cells and suggested possible dual treatment regimens. In addition, it has been reported that CLA can up-regulate the tumor suppressor gene PTPRG, and may have anti-cancer properties (Wang et al., 2006; Boni and Sorio, 2022). The studies showed that among CLA isomers which are t10, c12-CLA, and c9, t11-CLA were the most potent which have been implicated as a tumor suppressor gene in kidney and lung cancers. The results indicate that dietary CLA might serve as a chemo-preventive and chemo-therapeutic agent in human breast cancers by up-regulating the estrogen-regulated tumor suppressor gene (Thavasuraj et al., 2020), PTP gamma expression. Another team also found that mothers consuming mostly organic milk and meat products have higher levels of rumenic acid in their breast milk (Islam et al., 2008).

CLA and the long-chain polyunsaturated n−3 fatty acids have been shown in vivo and in vitro to reduce tumor growth. Tumor growth could occur by slowing or stopping cell replication (by interfering with transition through the cell cycle), increasing cell death (via necrosis and/or apoptosis), or both (Flis and Molik, 2021). The anticancer effects of fatty acids, shown in vivo, could also be mediated by effects on the host’s immune system (Field and Schley, 2004; Gorain, 2024).

CLA occurs naturally in dairy products but is also produced by certain strains of human intestinal bifidobacteria (Wang et al., 2023). The acid is known to exhibit potent anticancer effects both in vivo and in a range of tumor epithelial cell lines (HT-29 human colon cancer cell line) (Shafiqur 2006; Koronowicz and Banks, 2018). Elder et al. (2002) suggest that trans-10, cis-12 CLA has significant effects on the metabolism of essential fatty acids in HepG2 cells, whereas cis-9, trans-11 CLA does not have any effect in this respect.

This study aimed to evaluate the efficiency of milk-rich in CLA, fat containing CLA, commercial CLA, and pure CLA as anticancer (in vitro study). The study was preceded on different cancer cell lines (EACC, HeLa cells, HepG-2, MCF-7), and the anticancer activity and IC50 were estimated. The study was also conducted on experiment animals to investigate the administration of previous milk materials on the life span of tumor transplanted mice as an in vivo study.

MATERIALS AND METHODS

The Institutional Animal Care and Use Committee, the Ethics Committee of the Faculty of Veterinary Medicine, Cairo University, Giza, Egypt, accepted this study protocol (Approval No. VetCU10102019088).

Normal milk and milk rich in CLA were obtained from buffaloes fed on special diet supplemented with olive oil and sunflower mixture (1:1) with monensin to enhance the production of CLA.

Milk fat and fat-rich in CLA were extracted from each milk using an organic solvent (Chloroform : Methanol, 2/1), (Castro-Gómez et al., 2014)

Commercial CLA was purchased from MusclePharm, USA and diluted 10-fold by 1% DMSO while the pure CLA (Sigma-Aldrich) was prepared using 50 mg in ml of 1% DMSO.

Swiss albino female mice weighing 20-25 g were used for tumor transplantation.

Three cancer cell lines, HeLa, HepG-2, and MCF-7 were obtained from Vacsera Egypt. Ehrlich Ascites Carcinoma Cells (EACC) were obtained from National Cancer Institute, Egypt.

The anticancer activity of the tested samples was determined by specific techniques as follows: The viability of cancer cells (HeLa, HepG-2, and MCF-7) was examined with the neutral red assay (Retpetto et al., 2008). Cancer cell lines were cultured and tested for their viability after incubation with special media containing the tested materials (milk and CLA). The milk was added to the media as 10% in experiments of milk testing while in CLA testing fat extract containing CLA was added as that found on the 10% milk (1 mg/10⁶ cells). Standard and commercial CLA were added using their diluted solutions at the level in the 10% milk as mentioned.

The cytotoxicity parameter, IC₅₀ was calculated as moderate if IC₅₀ ranges from 1 μg/ml to 10 μg/ml; and weak for IC₅₀ higher than 10 μg/ml. The dose response curve of compounds was analyzed using E_max model (Eq. 1).

$\%Cellviability=(100-R\times \left(1-{\left(D\right)}^m/\left(K_d{}^m+Dm\right)\right)+R\dots \dots \dots \dots \dots \dots \dots \dots \dots \mathrm{..}$ (Eq.1)

Where R is the residual unaffected fraction (the resistance fraction), [D] is the drug concentration used, K_d is the drug concentration that produces a 50% reduction of the maximum inhibition rate and m is a Hill-type coefficient. IC₅₀ was defined as the drug concentration required to reduce fluorescence to 50% of that of the control (i.e., K_d = IC₅₀ when R=0 and E_max =100-R), (Retpetto et al., 2008). The cytotoxicity of the extracts was also tested against EACC by viability Trypan blue exclusion test as mentioned.

The enzymatic activity of the caspase-3 class of proteases in the treated cell lines was determined using Caspase-3 Colorimetric assay Kit (R&D systems, Inc.).

The EACC transplanted animals were used for life span evaluation. In the later study, 90 female mature mice were used in two experiments (45 mice in each) to investigate the effect of feeding pasteurized buffaloes milk rich in CLA or normal buffalo milk on life span. Milk was used for feeding the mice at a rate of 10% of the diet as a dry matter basis. In the first experiment, mice were injected with tumor cells at the commencement of feeding 3 diets to three groups (n=15/group). The 1^st group was fed a normal diet, the 2^nd group was fed a normal diet with normal milk, and the 3^rd group was fed a normal diet with milk rich in CLA (about 2 mg CLA/mice/day). In the second experiment, 3 groups of mice were fed the diets for 30 days, and then, the mice were transplanted with tumor cells and the feeding system was continued until the end of the experiment (60 days).

SPSS software version 25 was used for the statistical analysis. All findings reported were based on triplicate experiments (p < 0.05).

RESULTS AND DISCUSSION

Results in Table 1 and Fig. 1 show that the addition of normal milk and milk rich in CLA into the media of cancer cells had no inhibitory effects on cancer cells. The number of viable cells after incubation with milk enriched with CLA is nearly similar to untreated cells. In some cases, the milk samples gave high viability of cancer cells which seems that milk may enhance cancer cells’ growth due to its high dietary value in the media. However, the substitution of milk samples with their lipid fractions showed pronounced inhibitory effects on cancer cells’ growth. The action of the lipid fractions as an anticancer may be mainly due to the CLA and its isomers in these fractions. This result was confirmed when we added CLA either commercial or extracts to the media instead of milk, and the viability of cancer cells was inhibited. Similar inhibition trends were also observed on the 3 cell lines: Hela, HepG2, and MCF-7 (Table 2 and Fig. 2). This data also showed that the effect of CLA as an anticancer was concentration dependent.

Table 1
Anticancer activity of CLA on EACC cell line (in vitro study)

Tumor cells	Viable	Dead	anticancer %
Control	343	17	4.72
Normal Milk	365	13	3.43
Milk rich in CLA	355	11	3.00
Lipid extract of:
Normal milk	137	175	56.08
Milk rich in CLA	167	312	65.51
Standard CLA
CLA (Commercial)	26	219	89.87
Standard CLA	34	287	89.40

Figure 1
Anticancer activity of CLA on EACC cell line (in vitro study).

Table 2
Cytotoxicity of CLA on different cell lines

	IC50 (μl/ml)
	HeLa cell line	HepG2 cell line	MCF-7 cell line	EACC
Commercial CLA	1.6±0.5	7.6±1.5	1.2±0.	6.04±0.5
Standard CLA	2.7±4	4.2±10.1	3.6±5.3	2.61±2.1

Figure 2
Cytotoxicity of CLA on different cell lines. The mean ± SD (p<0.05).

Furthermore, the activity of caspase-3 was enhanced as shown in fig. 3 in cancer cells treated with samples containing CLA indicating that CLA supplementation enhances the apoptosis of cancer cells (Table 3).

Similar findings have been obtained when bovine milk fat was added to the diet which inhibited tumor cells’ growth and observed that the effect was mainly due to the conjugated linoleic acid (Ip et al., 1994c).

Table 3
Caspase-3 activity in EACC after incubation with different samples containing CLA

Treatment	Caspas-3 activity (OD405)
Control	0.445
Normal Milk	0.412
Milk rich in CLA	0.434
Lipid extract of Normal milk	0.862
Lipid extract of milk rich in CLA	0.932
CLA (Commercial)	1.231
Standard CLA	1.433

Figure 3
Caspas-3 activity in EACC after incubation with different samples containing CLA.

MCF-7 breast cancer cells were affected by CLA but with lower potential than the other cancer cells and this effect could be enhanced by Pluronic F127 as mentioned by Guo et al., (2007). The study showed that conjugated linoleic acid-coupled Pluronic F127 (Plu-CLA) enhances anticancer efficacy in MCF-7 cancer cells when compared to conjugated linoleic acid (CLA) itself. The authors also illustrated that the mechanism CLA was simply coupled to Pluronic F127 through ester linkage between the carboxyl group of CLA and hydroxyl one of Pluronic at melting state without solvent or catalyst. Plu-CLA significantly enhanced apoptosis with increasing concentration compared with CLA itself. Moreover, it was found that p53, p21, and Bax were up-regulated, whereas Bcl-2 and procaspase 9 were down-regulated with increasing concentrations of Plu-CLA. These results were attributed to the sensitization activity of Pluronic F127.

In our additional work, yogurt produced from milk rich in CLA and its isomers was used instead of milk, and a similar trend was observed (data not shown). The present results clearly show the anticancer activity of CLA-enriched milk and its products which can be used as functional foods. The usage of natural milk clotting enzymes, extracted from edible mushrooms such as Pleurotus albidus, could aid in the concentration of CLA increasing its ani-cancer effects (Abdel-Rahman et al., 2018).

Data in Fig. 4 show the life span of animal groups fed on normal milk and milk enriched with CLA. Using buffalo's milk rich in CLA in feeding female mice at a rate of 10% of daily diet intake improved the span of life. The animals have a longer life span and better health than the other two groups. The mice in the 2^nd experiment showed the same behavior as in the 1^st experiment, but the life span of the mice was longer than that of the first experiment (Fig. 5). The mortality rate after tumor transplantation was 100% for groups 1, 2, and 3 on days 13,16, and 23 respectively. The present study also showed that the life span of tumor transplanted mice was elongated in animals fed a diet containing milk enriched with CLA than those without CLA.

Figure 4
Effect of diets containing milk rich in CLA on life span of tumor transplanted mice.

Figure 5
Effect of diets containing milk rich in CLA on life span of tumor transplanted mice firstly fed on diet for 30 days before tumor transplantation.

The prolonged life span in tumor transplanted mice by milk enriched in CLA administration illustrates the relative inhibition of tumor growth when compared to the control. The inhibitory effect was more pronounced when animals were previously fed with CLA-enriched milk for 30 days before tumor transplantation and this may be due to the prophylactic effect of CLA in animals against cancer. This observation was previously illustrated by Liew et al., (1995) who reported the protection of conjugated linoleic acids against 2-amino-3-methylimidazole [4,5f] quinolone-induced colon carcinogenesis in the F344 rats. This protection may be due to the enhancement of the immune system in animals by CLA as mentioned by Field and Schley (2004).

In this concern, Ip et al. (1991, 1994b) illustrated that CLA and food containing this acid and its isomers gave cancer prevention in animals.

CLA when used separately or in lipid extracts has anticancer activity on different cell lines (in vitro study). It has been speculated that CLA may positively influence the tumor suppressor gene PTPRG associated with mammary tumors and may exhibit some anti-cancer properties in experimental animal models. In this concern, the study of Shfiqur (2006) indicated that the antiproliferative effect of CLA on HT-29 colon cancer cell line may be mediated by differentiation and apoptosis and by modulation of FAS and SCD activities. Also, the anticarcinogenic action of CLA stems from its ability to inhibit proliferation and promote differentiation in mammary epithelial cells.

Various studies have suggested that CLA may act by antioxidant mechanisms (Ha et al., 1990), pro-oxidant cytotoxicity (Schonberg and Krokan, 1995), inhibition of nucleotide and protein synthesis (Schultz et al., 1992), reduction of cell proliferative activity (Ip et al., 1994a) and inhibition of both DNA-adduct formation (Zu and Schut, 1992) and carcinogen activation (Liew et al.,1995).

Other candidate mechanisms for CLA action have been proposed for their immune effects, including changes in; 1) membrane structure and composition, 2) membrane-mediated functions and signals (e.g., proteins, eicosanoids), 3) gene expression, and 4) immune development (Field and Schley, 2004). In this respect, CLA modulates markers of immunity and eicosanoid formation in numerous species as well as lipid metabolism and gene expression. Also, mechanisms of inhibition of carcinogenesis may include reduction of cell proliferation, alterations in the components of the cell cycle, and induction of apoptosis (Belury, 2002; Joseph, 2020).

In addition to evidence showing that CLA may induce PPARγ-responsive genes in vivo, CLA may induce the level of PPARγ itself (Evans et al., 2000; Badawy et al., 2023). The authors explained that PPARγ2 is thought to be one of several transcription factors required for adipose tissue differentiation. Also, new evidence suggests that activators of PPARγ are protective against cancers arising in the mammary gland, colon, and prostate. These studies showed that perhaps the ability of PPARγ to mediate the effects of CLA is through increased levels of PPARγ protein and/or through transcription factors for adipose tissue differentiation.

It is worth mentioning that other unsaturated fatty acids in milk fat also enhance the inhibition activity against cancer cells. This effect is due to its activity to produce free radicals by lipid peroxidation and these radicals attack and kill cancer cells (Das et al., 1987a ,1987b). Gamma linolenic acid, (GLA or omega 3) showed a powerful effect as an anticancer on different types of cancer and gave antitumor on cultured human neuroblastoma cells (Fujiwara et al., 1994), has cytotoxic to 36B10 malignant rat astrocytoma cells (Vartak et al., 1989) and used as therapy of human gliomas (Bakshi et al., 2003).

CONCLUSION

CLA and its isomers when used separately or in lipid extracts as well as enriched milk have anticancer activity on different cell lines (in vitro study) and tumor transplanted animals (in vivo study). This group of fatty acids offers the possibility that several types of cancers in humans can be prevented with a diet rich in a diversity of chemo preventive compounds, including CLA and other unsaturated fatty acids. The action of CLA acid and its isomers is due to the enhancement of cancer cells to enter apoptotic pathways or positively influence specific genes such as tumor suppressor genes or that affect the cell life cycle of cancer cells. Our results indicate that the effects of CLA are more prophylactic rather than therapeutic. More work is required to fully understand the implications of dietary CLA and the possibility of lowering the risk for human cancer development.

ACKNOWLEDGMENT

The authors are thankful to the Deanship of Graduate Studies and Scientific Research at University of Bisha for supporting this work through the Fast-Track Research Support Program.

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Publication Dates

• Publication in this collection
  14 July 2025
• Date of issue
  Jul-Aug 2025

History

• Received
  21 Sept 2024
• Accepted
  25 Nov 2024