Withania somnifera modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC’s)

Background Cancer and inflammation are associated with cachexia. Withania somnifera (W. somnifera) possesses antioxidant and anti-inflammatory potential. We investigated the potential of an aqueous extract of the root of W. somnifera (WRE) to modulate cytokines, antioxidants and apoptosis in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC’s). Methods Cytotoxcity of WRE was determined at 24 and 72 h (h). Oxidant scavenging activity of WRE was evaluated (2, 2-diphenyl-1 picrylhydrazyl assay). Glutathione (GSH) levels, caspase (− 8, − 9, − 3/7) activities and adenosine triphosphate (ATP) levels (Luminometry) were thereafter assayed. Tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 levels were also assessed using enzyme-linked immunosorbant assay. Results At 24 h, WRE (0.2–0.4 mg/ml) decreased PBMC viability between 20 and 25%, whereas it increased THP-1 viability between 15 and 23% (p < 0.001). At 72 h, WRE increased PBMC viability by 27–39% (0.05, 0.4 mg/ml WRE) whereas decreased THP-1 viability between 9 and 16% (0.05–0.4 mg/ml WRE) (p < 0.001). Oxidant scavenging activity was increased by WRE (0.05–0.4 mg/ml, p < 0.0001). PBMC TNF-α and IL-10 levels were decreased by 0.2–0.4 mg/ml WRE, whereas IL-1β levels were increased by 0.05–0.4 mg/ml WRE (p < 0.0001). In THP-1 cells, WRE (0.05–0.4 mg/ml) decreased TNF-α, IL-1β and IL-6 levels (p < 0.0001). At 24 h, GSH levels were decreased in PBMC’s, whilst increased in THP-1 cells by 0.2–0.4 mg/ml WRE (p < 0.0001). At 72 h, WRE (0.1–0.4 mg/ml) decreased GSH levels in both cell lines (p < 0.0001). At 24 h, WRE (0.2–0.4 mg/ml) increased PBMC caspase (-8, -3/7) activities whereas WRE (0.05, 0.1, 0.4 mg/ml) increased THP-1 caspase (-9, -3/7) activities (p < 0.0001). At 72 h, PBMC caspase (-8, -9, -3/7) activities were increased at 0.05–0.1 mg/ml WRE (p < 0.0001). In THP-1 cells, caspase (-8, -9, -3/7) activities and ATP levels were increased by 0.1–0.2 mg/ml WRE, whereas decreased by 0.05 and 0.4 mg/ml WRE (72 h, p < 0.0001). Conclusion In PBMC’s and THP-1 cells, WRE proved to effectively modulate antioxidant activity, inflammatory cytokines and cell death. In THP-1 cells, WRE decreased pro-inflammatory cytokine levels, which may alleviate cancer cachexia and excessive leukaemic cell growth. Electronic supplementary material The online version of this article (10.1186/s12906-018-2192-y) contains supplementary material, which is available to authorized users.


Background
Chronic inflammation plays an essential role in malignancies [1] through the initiation, promotion and progression of tumours [2]. Usually, the host-mediated anti-tumour activity overcomes the tumour-mediated immunosuppressive activity leading to the elimination of cancerous cells [2]. However, in the presence of an inadequate host antitumour defence, the pro-inflammatory tumour microenvironment is enhanced and promotes tumour development, invasion, angiogenesis and metastasis [2].
The cachectic syndrome is prominent in malignancies occurring in up to 50% of all cancer patients [3]. It is a progressive, debilitating condition leading to abnormal weight loss, as a result of adipose tissue (85%) and skeletal muscle (75%) depletion [3][4][5]. Modulation of lipogenesis and lipolysis is essential in maintaining adipose tissue mass. Lipoprotein lipase (LPL) hydrolyses fatty acids (FA's) from plasma lipoproteins, thereafter FA's are transported to adipose tissue for triacylglycerol (TAG) production, whereas hormone sensitive lipase (HSL) hydrolyses TAG's into FA's and glycerol [3]. Literature shows that decreased serum LPL levels/activity and increased HSL levels/activity are associated with cachexia [6][7][8]. Additionally, increased proteolysis [9] and decreased proteogenesis has been established in cachectic patients [10]. The ATP-ubiquitin-dependent proteolytic pathway has been shown to be responsible for the accelerated proteolysis seen in a variety of wasting conditions, including cancer cachexia [11].
Cancer patients suffer from a wide range of side-effects caused by current cancer chemotherapeutic and radiotherapeutic agents. Patients are constantly seeking alternative traditional remedies to alleviate their discomfort. Withania somnifera (L.) Dunal (W. somnifera) is a well known medicinal plant cultivated in India, parts of East Asia and Africa [31]. It is commonly referred to as Ashwagandha and belongs to the Solanaceae family [31]. Compounds isolated from W. somnifera include withaferin A and 3-β-hydroxy-2, 3 dihydro withanolide F [32]. The major constituent of the root extract of W. somnifera is withanolide-A [33]. W. somnifera is frequently used in Ayurvedic medicine due to its various medicinal properties [31]. These properties include anti-inflammatory [34], antioxidant and immunemodulatory activities [35]. W. somnifera was found to be an immune regulator in inflammation animal models [36]. The immunosuppressive action of W. somnifera may be due to the presence of withanolides, steroidal lactones and a few flavanoids [37]. In addition, W. somnifera formulation (WSF) has shown anti-proliferative potential in human promyelocytic leukemia (HL-60) cells, by activating the intrinsic and extrinsic apoptotic pathways [38]. When used together, W. somnifera formulations aid the host to effectively fight cancer and reduce the harmful effects of chemotherapy and radiotherapy [39].
There is a need for the discovery of an inexpensive cancer cachectic treatment to improve the prognosis of cancer patients and to establish a mechanism of regulation of the immune system, inflammasome and apoptosis in order to prevent/decelerate the rapid depletion of skeletal muscle and adipose tissue. We investigated the effect of an aqueous extract of the root of W. somnifera (W RE ) on antioxidant capacity, inflammatory cytokine levels and cell death induction in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC's).

Plant extraction
The roots of W. somnifera were dried and milled before being sequentially extracted in ethanol and distilled water. Ethanol (200-350 ml) was added to the milled root (10-30 g) and extracted overnight by shaking (4×g, 37°C). The ethanol extracts were thereafter filtered, evaporated using a rotary evaporator, dried (37°C) and stored (4°C). The root material was thereafter extracted with distilled water (200-350 ml) by shaking (4×g, 75°C) for a period of 6 hours (h). Water extracts were filtered, dried and stored (4°C).
Tissue culture THP-1 cells were grown in the appropriate tissue culture conditions in a 75 cm 3 tissue culture flask (37°C, 5% CO 2 ). The growth media comprised of RPMI-1640, FCS (10%) and PSF (2%). Cells were thawed, seeded into a 75 cm 3 tissue culture flask at a concentration of 3 × 10 5 cells/ml and incubated (37°C, 5% CO 2 ). THP-1 cells were allowed to grow for 2-3 days before the cells were centrifuged (162×g, 10 min) and re-suspended in fresh growth media. The number of cells should not exceed 8 × 10 5 cells/ml, therefore the cells/ml was quantified daily by trypan blue staining. Once the cell count reached 8 × 10 5 cells/ml the THP-1 cells were split/diluted to 3 × 10 5 cells/ml with media and incubated. Subsequent experiments were conducted once the cell numbers were sufficient.

The glutathione assay
The GSH-Glo™ assay (Promega, Madison, USA) was performed to measure GSH levels. Standard GSH solutions were prepared by diluting a 5 mM stock solution serially (1.56-50 μM) and PBS (0.1 M) was the standard blank. Cells (50 μl/well, 2 × 10 5 cells/ml) and standards were added into an opaque 96-well plate, followed by GSH-Glo™ reagent (25 μl/well) and allowed to incubate (30 min, RT) in the dark. Luciferin detection reagent (50 μl/well) was subsequently added and plates incubated (15 min, RT) in the dark. The absorbance was read on a Modulus™ microplate luminometer (Turner Biosystems, Sunnyvale, USA) and GSH concentrations were calculated by extrapolation from a standard curve.

Statistical analysis
Statistical analysis was performed using the STATA and GraphPad Prism statistical analysis software. The one-way analysis of variance (ANOVA) was used to compare between groups, followed by the Tukey multiple comparisons test, with p < 0.05 defining statistical significance.

Results
The oxidant scavenging potential of W RE The oxidant scavenging activity of W RE using the DPPH assay is shown in Fig. 1. W RE (0.05-0.4 mg/ml) significantly increased DPPH scavenging activity by 13.33-46. 38% (Fig. 1, p < 0.0001).

The antioxidant potential of W RE
The endogenous antioxidant activity of W RE was determined by measuring GSH levels in both cell lines (  Table 2, p < 0.0001). PBMC caspase-9 activity was increased by 0.05 and 0.2 mg/ml W RE but decreased by 0.1 and 0.4 mg/ml W RE relative to the control ( Table 2, p < 0.0001). In PBMC's, the increased caspase activity may be related to the decreased GSH levels at 24 h. A decrease in GSH levels may allow for an increase in ROS levels which can activate apoptotic pathways. Caspase-3/7 activity was increased in PBMC's by 0.05-0. 4 mg/ml W RE compared to the control ( Table 2, p < 0. 0001), suggesting an increased execution of apoptotic cell death. In azoxymethane-induced colon cancer in mice, W. somnifera has been shown to modulate TCA cycle enzymes and the electron transport chain [41]. The PBMC ATP levels were increased by 0.1, 0.4 mg/ml W RE but decreased by 0.05, 0.2 mg/ml W RE compared to the control ( Table 2, p < 0.0001), which may be related to the modulation of the electron transport chain by W. somnifera. W RE pro-apoptotic effects in THP-1 cells treated for 24 h are shown in Table 3. At 24 h, THP-1 caspase-9 activity was decreased by 0.2 mg/ml W RE but increased by 0. 05, 0.1, 0.4 mg/ml W RE compared to the control (Table 3, p < 0.0001). At 0.2 mg/ml W RE , the decreased caspase-9 activity may be related to the increased GSH levels. An increase in GSH levels may decrease ROS levels thus minimising mitochondrial depolarisation and the activation of the intrinsic apoptotic pathway. In THP-1 cells, W RE (0.05-0.4 mg/ml) decreased caspase-8 activity, whereas increased caspase-3/7 activity and ATP levels relative to the control (Table 3, p < 0.0001). Elevated caspase (-9, -3/7) activities suggests the initiation of the mitochondrial apoptotic pathway.
The pro-apoptotic effect of W RE in PBMC's treated for 72 h is shown in Table 4. At 72 h, PBMC caspase-8 activity was increased by 0.05-0.2 mg/ml W RE but decreased by 0.4 mg/ml W RE compared to the control ( Table 4, p < 0.0001). PBMC caspase-9 activity was increased by 0.05-0.1 mg/ml W RE but decreased by 0.2-0. 4 mg/ml W RE relative to the control (Table 4, p < 0. 0001). In PBMC's, caspase-3/7 activity was increased by 0.05, 0.1, 0.4 mg/ml W RE whereas it decreased by 0. 2 mg/ml W RE compared to the control (Table 4, p < 0. 0001). At 0.05-0.1 mg/ml W RE , the increased caspase-3/ 7 activity is consistent with the significantly increased caspase -8 and -9 activity. At 0.2 mg/ml W RE , caspase-8 activity was minimally increased and caspase-9 activity significantly decreased which lead to the decreased caspase-3/7 activity. At 0.4 mg/ml W RE , although both caspase -8 and -9 activities were decreased, caspase-3/7 activity was increased. A previous study has demonstrated that one activated executioner caspase can cleave and activate other executioner caspases resulting in positive feedback loop of caspase activation [42] which may account for the increased caspase-3/7 activity at 0.4 mg/ml W RE . W RE (0.05-0. 4 mg/ml) decreased PBMC ATP levels relative to the control (Table 4, p < 0.0001). W RE pro-apoptotic effects in THP-1 cells treated for 72 h are shown in Table 5. At 72 h, THP-1 caspase (-8, -9, -3/7) activity and ATP levels were increased by 0.1-0. 2 mg/ml W RE as compared to the control ( Table 5, p < 0. 0001), suggesting an increase in THP-1 apoptotic cell death. THP-1 caspase (-8, -9, -3/7) activity and ATP levels were decreased by 0.05, 0.4 mg/ml W RE relative to the control (Table 5, 72 h, p < 0.0001), suggesting a decrease in THP-1 apoptosis.

Discussion
Cachexia patients experience excessive weight loss due to increased lipolysis and proteolysis which have been linked to elevated levels of pro-inflammatory cytokines, oxidative stress and apoptosis [3,5,30]. Previously, the powdered root of W. somnifera displayed immune modulatory properties [43] and WSF has been shown to increase caspase-3 activity, subsequently inducing apoptosis [38]. The objective of this study was thus to investigate the modulation of cytokines, antioxidants and cell death by W RE in PBMC's and THP-1 cells. Dhanani et al. (2017) showed that the root extract of W. somnifera inhibited 50% of DPPH at a concentration of 0.4 mg/ml [44]. Our results indicated that W RE has oxidant scavenging potential ranging between 13 and 46% at 0.05-0.4 mg/ml. ROS plays an essential role in tumour initiation, inflammation, protein degradation and apoptosis. The antioxidant potential of W RE may decrease inflammatory cytokine levels as well as ROS induced apoptosis.
Pro-inflammatory cytokines, over a longstanding time period, stimulate the production of genotoxic molecules [nitric oxide (NO), ROS] and tumour progression by promoting angiogenesis and metastasis [1,2]. In addition, pro-inflammatory cytokines activate NF-κB which regulates the expression of genes involved in the suppression of tumour apoptosis, stimulation of tumour cell cycle progression and enhancement of inflammatory mediators [1,2]. NF-κB promotes tumour progression, invasion, angiogenesis and metastasis [1,2].
Previous literature has shown that IL-1 stimulates growth and invasion of malignant cells [2]. Additionally, IL-6 has been shown to target cell cycle progression and anti-apoptotic genes leading to tumour proliferation and anti-apoptotic potential [2]. The ability of W RE to increase pro-inflammatory cytokines such as IL-1β in PBMC's may aid in cancerous cell elimination through increased host anti-tumour activity. Conversely, in THP-1 cells, the decrease in TNF-α, IL-6 and IL-1β levels by W RE may prevent excessive activation of NF-κB, diminish cytokine induced tumour immunosuppressive activity and cancer progression.
With regard to cancer cachexia, IL-6 decreased LPL activity in adipose tissue of mice [22] and IL-1 directly modulates lipid metabolism by suppressing LPL activity [23]. TNF-α decreased LPL activity in adipose tissue of human (maintained in organ culture), rat, mouse, and guinea pigs [21]. Additionally, TNF-α inhibits the production of LPL and reduces the rate of LPL gene transcription in mouse 3 T3-L1 adipocytes, hence preventing the formation of new lipid stores while stimulating HSL and increasing lipolysis [3,20,46]. The potential of W RE to decrease proinflammatory cytokine levels in PBMC's and THP-1 cells suggests a decrease in LPL inhibition and HSL stimulation, thus maintaining lipogenesis and minimizing lipolysis. IL-6 and TNF-α further contribute to cachexia by stimulating muscle catabolism via the activation of proteasome pathways [24,25,47]. In cachexia, NF-κB activation induces ubiquitin-proteasome pathway activity and suppresses MyoD expression [48], thereby increasing proteolysis and reducing muscle replenishment [49]. By decreasing TNF-α and IL-6 levels in PBMC's and THP-1 cells, W RE may prevent excessive activation of NF-κB and proteasome pathways, ultimately decreasing proteolysis associated with the cachectic syndrome. Taken together, W RE may be able to decrease tissue wasting through the down regulation of pro-inflammatory cytokine levels.
The immunosuppressive and anti-inflammatory cytokine, IL-10, inhibits tumour development, tumour progression, modulates apoptosis and suppresses angiogenesis during tumour regression [1,2]. Additionally, IL-10 inhibits NF-κB activation and subsequently inhibits pro-inflammatory cytokine production (TNF-α, and IL-6) [2]. In PBMC's and  THP-1 cells, the decreased IL-10 levels may be due to IL-10 combating increased pro-inflammatory cytokines levels (TNF-α, IL-6, IL-1β). Antioxidants protect cells from increased oxidative stress [50]. GSH is a potent antioxidant that effectively scavenges ROS both directly and indirectly [50]. W. somnifera has previously been shown to possess chemo-preventive activity which may be a consequence of its antioxidant capacity [39]. The 24 h results showed that W RE decreased GSH levels in PBMC's, whereas it increased GSH levels in THP-1 cells. However, at 72 h, W RE decreased GSH levels in both cell lines. Notably, GSH levels (72 h) were higher in control PBMC's (4.79 μM) compared to control THP-1 cells (1.61 μM), suggesting a higher oxidant defence in PBMC's.
Increased caspase-3 activity, proteasome activity and E3 ubiquitin-conjugating enzyme expression is associated with increased proteolysis [51]. Therefore the ability of W RE (0.4 mg/ml, 72 h) to down regulate caspase activity in PBMC's and THP-1 cells may decrease proteolysis and the progression of cancer cachexia.
A successful anti-cancer drug should kill or incapacitate cancer cells without causing excessive damage to normal cells [39]. The potential of W RE to regulate PBMC apoptosis while increasing cancerous THP-1 cell apoptosis may be beneficial to cancer patients by preventing excessive cancerous cell growth while minimally effecting healthy PBMC's.  (Values expressed as mean ± SD, ** p < 0.005, *** p < 0.0001 compared to the control)