Plant derivatives have great promise as anticancer agents. In this study, low concentrations of fenugreek extract was found to induce death of Jurkat cells, a commonly used cell line model to understand anticancer functions and underlying mechanisms of exploratory drugs or compounds. Fenugreek extract affected the viability of Jurkat cells in a dose- and time-dependent manner. Fenugreek extract-induced death was preceded by dramatic morphological changes involving the appearance of multiple large vacuoles and transcriptional up-regulation of LC3, both of which imply activation of autophagy. Results from this study imply autophagy as a novel mechanism by which fenugreek may bring about death of preneoplastic/neoplastic cells. T-cell lymphoid malignancies are a group of highly aggressive diseases that are generally resistant to current treatment modalities and results from this study indicate benefits of fenugreek as a potential therapeutic. Further, induction of autophagy may be a general anticancer mechanism underlying chemopreventive/therapeutic function of fenugreek.
Anticancer properties of fenugreek or its constituents involve multiple functional and molecular targets. Fenugreek induced apoptosis in a wide variety of tumor cell lines, including those of human colon
[31, 32], and liver
. Fenugreek blocked migration and invasion by reducing matrix metalloproteinase expression in human prostate cancer PC-3 cells
, decreased nitric oxide (NO) and prostaglandin production by suppressing iNOS and COX-2, respectively, in an osteosarcoma cell line
. Fenugreek blocked activation of NF-KB, I kappa B kinase and AKT and suppressed the production of various proinflammatory cytokines like IL-6, IL-1 and TNF-α by cancer cells
[31, 36]. An earlier study noted the formation of cytoplasmic vacuoles in breast tumor tissue of 7, 12-dimethylbenz (α) anthracene (DBMA) treated rats upon feeding of fenugreek compared to control
. To our knowledge, ours is the first report to show death of human cancer cells by fenugreek-induced autophagy.
Apoptosis and autophagy are important pathways designed to cause programmed death of stressed cells and cancers suppress both these pathways and evade death. Autophagy has been defined as a process of transport of cytoplasmic contents to the lysozomes in double membrane compartments, called vacuoles
. Autophagy is a physiological response to stress and has been suggested to enable cells to adapt and survive and, hence, is considered a pro-survival mechanism
. Autophagy has also been defined as a highly conserved process of programmed cell death
 and its activation causes cell death
. Defective regulation of autophagy in cancers suggests that autophagy is a true tumor-suppressor pathway
[41, 42] and this is further supported by the fact that several commonly activated oncogenes (for example, those encoding PI3K, TOR, Bcl-2) inhibit autophagy, whereas commonly mutated or epigenetically silenced tumor suppressor genes (such as those encoding p53, PTEN, TSC1/TSC2) stimulate autophagy
Several studies have used electron microscopic methods to detect autophagic vacuoles. However, fenugreek-induced vacuoles in Jurkat cells were large and distinct and could be directly seen using an inverted light microscope. This study established the vacuolization as an autophagic process from the transcriptional up-regulation LC3. LC3 plays a critical role in autophagy and is considered suitable marker of this process
. In the present study, fenugreek extract increased LC3 transcript level with increasing concentrations of fenugreek extract over a period of 48 h. Harmol, a beta carotenoid, induced death of lung cancer cells A549 was associated with increased expression of LC3
 and siRNA knockdown of LC3 resulted in blockade of cell death, which, in turn, served to prove that harmol-induced autophagy was only a death mechanism
. Transcripts of beclin 1 and ATG5, the other autophagic pathway genes, did not show any decline in our fenugreek-treated Jurkat cells and this was similar to harmol-treated A549 cells which failed to show any changes in the expression and phosphorylation of beclin 1
Some health benefits of various medicinal plants or their constituents have been attributed to their ability to induce autophagy in diseased cells
. Experiments performed to measure the autophagy-inducing ability of several plant constituents, for eg., fisetin (found in tomatoes, apples, onions, and grapes), genistein (soybean) and quercetin (apple skins and red onions), report opposing findings (induction or suppression) resulting in reduced clarity on the beneficial effect of autophagy in cancer treatment. These contradictory results have been attributed to several factors, such as different methods employed to measure autophagy and the use of different cell types, such as cancerous cells versus normal cells
. Also, autophagy, at low levels, was suggested to serve a pro-survival role in tumors
. However, results from our study revealed increased production of autophagic markers and decreased viability to be associated with increasing concentrations of fenugreek implying only an anti-proliferative /pro-death function for autophagy at all concentrations of fenugreek extract. Results from our study support the notion that fenugreek-induced autophagy in Jurkat cells could only be an antitumor function.
Some of the components of fenugreek extract identified by GC-MS in our study have already been shown to possess significant anticancer activities and these include gingerol
, and eugenol
[51–53]. Future studies will be designed to identify the specific compound(s) responsible for induction autophagy.
The cytotoxic effects of fenugreek are limited, mostly, to cancer cells
[25, 32]. Bioavailability of therapeutic components of fenugreek, unlike curcumin, is high as seen from its hepatoprotective effect from various in vivo studies
[54, 55]. Feeding studies have indicated tolerance and absence of any toxicities of up to several grams of fenugreek per day over several weeks
The authors acknowledge some limitations. The study included a limited type and number of cell lines and lacked specific assays to confirm apoptosis and autophagy. Nevertheless, the present study identified autophagy as a new mechanism by which fenugreek may exert its therapeutic/chemoprevention/anticancer function.