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

Background Cancer cachexia is associated with increased pro-inflammatory cytokine levels. Centella asiatica (C. asiatica) possesses antioxidant, anti-inflammatory and anti-tumour potential. We investigated the modulation of antioxidants, cytokines and cell death by C. asiatica ethanolic leaf extract (CLE) in leukaemic THP-1 cells and normal peripheral blood mononuclear cells (PBMC’s). Methods Cytotoxcity of CLE was determined at 24 and 72 h (h). Oxidant scavenging activity of CLE was evaluated using the 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. Glutathione (GSH) levels, caspase (−8, −9, −3/7) activities and adenosine triphosphate (ATP) levels (Luminometry) were then assayed. The levels of tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 were also assessed using enzyme-linked immunosorbant assay. Results CLE decreased PBMC viability between 33.25–74.55% (24 h: 0.2–0.8 mg/ml CLE and 72 h: 0.4–0.8 mg/ml CLE) and THP-1 viability by 28.404% (72 h: 0.8 mg/ml CLE) (p < 0.0001). Oxidant scavenging activity was increased by CLE (0.05–0.8 mg/ml) (p < 0.0001). PBMC TNF-α and IL-10 levels were decreased by CLE (0.05–0.8 mg/ml) (p < 0.0001). However, PBMC IL-6 and IL-1β concentrations were increased at 0.05–0.2 mg/ml CLE but decreased at 0.4 mg/ml CLE (p < 0.0001). In THP-1 cells, CLE (0.2–0.8 mg/ml) decreased IL-1β and IL-6 whereas increased IL-10 levels (p < 0.0001). In both cell lines, CLE (0.05–0.2 mg/ml, 24 and 72 h) increased GSH concentrations (p < 0.0001). At 24 h, caspase (−9, −3/7) activities was increased by CLE (0.05–0.8 mg/ml) in PBMC’s whereas decreased by CLE (0.2–0.4 mg/ml) in THP-1 cells (p < 0.0001). At 72 h, CLE (0.05–0.8 mg/ml) decreased caspase (−9, −3/7) activities and ATP levels in both cell lines (p < 0.0001). Conclusion In PBMC’s and THP-1 cells, CLE proved to effectively modulate antioxidant activity, inflammatory cytokines and cell death. In THP-1 cells, CLE decreased pro-inflammatory cytokine levels whereas it increased anti-inflammatory cytokine levels which may alleviate cancer cachexia.


Background
The role of inflammation in carcinogenesis has been extensively documented [1]. Although inflammatory responses have shown beneficial effects in tissue repair and pathogen elimination [1,2], chronic inflammation has been implicated in tumour initiation, promotion and progression [3]. During ideal conditions, the hostmediated anti-tumour activity combats the tumourmediated immunosuppressive activity and cancerous cells are sentenced to cell death [3]. In the event that the host anti-tumour activity is weakened/inadequate, the persistent and enhanced pro-inflammatory tumour microenvironment will facilitate tumour development, invasion, angiogenesis and metastasis [3].
Many malignancies are associated with the cachectic syndrome [4], a disorder characterised by abnormal weight loss [5] due to adipose tissue (85%) and skeletal muscle (75%) depletion [6]. The enzyme lipoprotein lipase (LPL) hydrolyses fatty acids (FA's) and transports FA's into adipose tissue for triacylglycerol (TAG) production, whereas hormone sensitive lipase (HSL) breaks down TAG's into FA's and glycerol [6]. Studies have revealed that decreased serum LPL levels/activity [7,8] and increased HSL levels/activity are associated with cachexia [9]. Additionally, increased proteolysis and decreased proteogenesis have been reported in cachectic patients [10]. The ATP-ubiquitin-dependent proteolytic pathway has been shown to be responsible for the excessive proteolysis seen in cancer cachexia [11].
Oxidative stress, inflammatory cytokines and apoptosis play a pivotal role in the initiation and development of cancer cachexia [12]. Inflammatory cytokine production is increased by lipopolysaccharide (LPS) potently stimulating macrophages [13]. The LPS signal is transduced by LPS binding to LPS binding protein, delivered to CD14 and transferred to Toll like receptor-4 [14]. This subsequently activates nuclear factor kappa B (NF-κB), which regulates the transcription of genes associated with inflammation, proliferation, invasion, angiogenesis and apoptosis [1,[15][16][17]. Previously, IL-1 [18], IL-6 (mice) [19] and TNF-α (rat, mouse and guinea pigs) [20] were shown to decrease LPL activity in adipose tissue. Decreased LPL activity reduces the uptake of exogenous lipids by adipose tissue [20], which decreases lipogenesis. Additionally, previous literature showed that TNF-α increased ubiquitin (concentrations and mRNA), while IL-6 increased the 26S proteasome and cathepsin activities, suggesting the activation of proteolytic pathways [21][22][23][24]. The activation of proteolytic pathways causes extensive muscle wasting through proteolysis. Taken together, an excessive increase in pro-inflammatory cytokine levels may increase tumour immunosuppressive activity [3], as well as tissue wasting [6].
Oxidative stress has been associated with tumour initiation, inflammation [2,3] and muscle wasting [25]. However, antioxidants have been shown to decrease muscle wasting by neutralizing reactive oxygen species (ROS) [1,25]. Elevated ROS levels activate apoptotic pathways, ultimately activating caspase-3 [26]. The activation of caspase-3 plays an important role in the execution of apoptosis as well as muscle proteolysis [27]. Additionally, in weight-losing upper gastrointestinal tract cancer patients, deoxyribonucleic acid (DNA) fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage were increased, whereas MyoD protein was decreased [6], suggesting increased apoptosis and decreased muscle replenishment.
There is a constant need for alternative traditional medicines to improve the prognosis of cancer patients and prevent chemotherapy and radiotherapy induced discomfort. The tropical medicinal plant Centella asiatica (Linnaeus) Urban (C. asiatica) is native to India, China, and South Africa [28]. It belongs to the Apiaceae family and is commonly referred to as Gotu kola, Asiatic pennywort and Tiger herb [28]. C. asiatica is widely used in Ayurvedic and Chinese traditional medicines due to its various medicinal properties. These properties include its hepato-protective, cardioprotective, anti-diabetic, antioxidant, anti-inflammatory and anti-tumour potential [28]. The major active compounds in C. asiatica are triterpene saponosides such as asiatic acid, madecassic acid and asiaticoside [28]. C. asiatica also contains flavonoid derivatives, vitamins, minerals, polysaccharides, sterols and phenolic acids [28]. C. asiatica has previously been used in treatment of inflammation due to its promising anti-inflammatory effects [29,30]. Additionally, C. asiatica extracts have demonstrated high antioxidant [31,32] and anti-proliferative activity in many cancerous cell lines [33].
There is a need for the discovery of an inexpensive cancer cachectic treatment. The ability of a plant extract to regulate inflammatory cytokines and cell death may elevate cancerous cell death and diminish tissue wasting. We investigated the potential of a C. asiatica ethanolic leaf extract (C LE ) to modulate inflammatory cytokines, antioxidants and cell death in leukaemic THP-1 cells and normal peripheral blood mononuclear cells (PBMC's).

Plant description and extraction
The plants official name is Centella asiatica (L.) Urb and has been confirmed by using the plant list [34]. The English name is Tiger herb. C. asiatica leaves were dried and milled. Ethanol (200-350 ml) was added to milled plant material (10-30 g) and extracted overnight by shaking (4×g, 37°C). Ethanol extracts were filtered, rotor evaporated, dried (37°C) 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 PS (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.

Glutathione assay
The GSH-Glo™ assay (Promega, Madison, WI, USA) was used 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 (duplicate wells), followed by GSH-Glo™ reagent (25 μl/well) and allowed to incubate (30 min, RT) in the dark. Subsequently, luciferin detection reagent (50 μl/well) was 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 (v5) statistical analysis software. The one-way analysis of variance (ANOVA) was used to make comparisons between groups, followed by the Tukey multiple comparisons test, with p < 0.05 indicating significant results.

Results
The oxidant scavenging potential of C LE The oxidant scavenging activity of C LE using the DPPH assay is shown in Fig. 1. C LE (0.05-0.8 mg/ml) significantly increased DPPH scavenging activity by approximately 45-84% (Fig. 1, p < 0.0001).

The antioxidant potential of C LE
The endogenous antioxidant activity of C LE was determined by measuring GSH levels in both cell lines (Table  1). At 24 h, GSH levels in PBMC's were increased by 0.05-0.2 mg/ml C LE but decreased by 0.4-0.8 mg/ml C LE relative to the control (Table 1, p < 0.0001). In THP-1 cells, C LE (0.05-0.8 mg/ml) increased GSH levels as compared to the control (Table 1, Table 3. At 24 h, C LE (0.05-0.8 mg/ ml) increased THP-1 caspase-8 activity as compared to the control ( Table 3, p < 0.0001). In THP-1 cells, caspase-9 activity and ATP levels were decreased by 0.05-0.4 mg/ml C LE, whereas increased by 0.8 mg/ml C LE relative to the control (Table 3, 24 h, p < 0.0001). The THP-1 caspase-3/7 activity was decreased by 0.2-0.4 mg/ml C LE, whereas increased by 0.05 and 0.8 mg/ml C LE as compared to the control (Table 3, 24 h, p < 0.0001). THP-1 caspase (−8, −9, −3/7) activities was increased by 0.8 mg/ml C LE , suggesting an increased initiation and execution of THP-1 apoptosis.
The pro-apoptotic effect of C LE in PBMC's treated for 72 h is shown in Table 4. At 72 h, PBMC caspase-8 activity was increased by 0.4 mg/ml C LE, whereas decreased by 0.05, 0.2, 0.8 mg/ml C LE relative to the control (Table 4, p < 0.0001). C LE (0.05-0.8 mg/ml) decreased PBMC caspase (−9, −3/7) activities and ATP levels as compared to the control (Table 4, 72 h, p < 0.0001). Decreased PBMC caspase activity suggests a decrease in PBMC apoptotic cell death.

Discussion
Cancer and cachexia have been associated with increased levels of oxidative stress, pro-inflammatory cytokines and apoptosis [6,27]. The medicinal plant, C. asiatica possesses anti-inflammatory [29] and anti-tumor activity [35], which can be beneficial in the treatment of cancer cachexia.
Previously, Zainol et al. (2003) reported that C. asiatica possessed antioxidant potential, possibly associated with phenolic compounds [36]. The DPPH assay revealed that C LE has oxidant scavenging potential ranging between 45 and 84% at 0.05-0.8 mg/ml C LE . ROS plays a pivotal role in tumour initiation, inflammation, protein degradation and apoptosis. The significant oxidant scavenging potential of C LE may decrease inflammatory cytokine levels and ROS induced apoptosis.
Inflammatory cytokines play an essential role in tumourgenesis and the cachectic syndrome [6]. Previously, Punturee et al. (2004) reported that C. asiatica ethanolic extract modulated/suppressed TNF-α production in mouse macrophages [39]. Our results also show that C LE decreased TNF-α concentration in PBMC's. Yun et al. (2008) reported that the pre-treatment of RAW264.7 cells with asiatic acid significantly reduced IL-6 production with minimal effects on TNF-α and IL-1β levels [37]. Our findings, however, suggest that C LE modulates proinflammatory cytokine levels. In both PBMC's and THP-1 cells, IL-1β and IL-6 levels were increased by the lower 0.05 mg/ml C LE concentration but decreased at the higher 0.4 mg/ml C LE concentration. Pro-inflammatory cytokines, over a chronic time period, stimulate the production of genotoxic molecules [nitric oxide (NO), ROS] and tumour progression by promoting angiogenesis and metastasis (Values expressed as mean ± SD, ** p < 0.001, *** p < 0.0001 compared to the control) [1,3]. Previous literature has shown that IL-1 stimulates malignant cell growth and invasiveness [3]. In addition, IL-6 exerts its tumour proliferative and antiapoptotic potential by targeting genes involved in cell cycle progression and the suppression of apoptosis [3]. The ability of C LE 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 IL-6 and IL-1β concentrations by C LE may diminish cytokine induced tumour immunosuppressive activity and cancer progression.
With regard to the cachectic syndrome, TNF-α inhibits the production of LPL and reduces the rate of LPL gene transcription [40][41][42], thereby preventing the formation of new lipid stores while stimulating HSL and increasing lipolysis [43]. In adipose tissue (in vivo), IL-6 decreased LPL activity leading to tissue wasting in cachectic individuals [19]. The potential of C LE (0.4 mg/ml) to decrease IL-6 and IL-1β concentrations in PBMC's and THP-1 cells suggests a decrease in LPL inhibition and HSL stimulation, thus contributing to lipogenesis maintenance and minimal lipolysis. IL-6 and TNF-α further contribute to cachexia by stimulating muscle catabolism through the activation of the ubiquitinproteasome pathway [21,22,44]. Furthermore, proinflammatory 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 [1,3]. Taken together, NF-κB promotes tumour progression, invasion, angiogenesis and metastasis [1,3]. In cachexia, NF-κB activation induces ubiquitin-proteasome pathway activity and suppresses MyoD expression [45], thereby increasing proteolysis and decreasing muscle replenishment [46]. By decreasing IL-6 and IL-1β concentrations in PBMC's and THP-1 cells, C LE (0.4 mg/ml) may prevent excessive activation of NF-κB and proteasome pathways, ultimately decreasing proteolysis. Taken together, C LE 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 [1,3]. Additionally, IL-10 inhibits NF-κB activation and subsequently inhibits proinflammatory cytokine production (TNF-α, and IL-6) [3]. With regard to tissue wasting, increased IL-10 levels in colon 26-bearing mice was reported to reverse the cachectic syndrome [47]. The decreased PBMC IL-10 concentration may be due to IL-10 combating increased pro-inflammatory cytokine levels (IL-6 and IL-1β). In THP-1 cells, the potential of C LE to increase IL-10 levels will facilitate a decrease in pro-inflammatory cytokine levels, a decrease in malignant cell progression and possibly alleviate the cancer cachectic syndrome.
At 72 h, caspase activities were decreased in both cell lines, suggesting a decreased activation of apoptosis. In PBMC's and THP-1 cells, the increase in GSH levels and the decrease in caspase (−9, −3/7) activities by C LE (0.05-0.8 mg/ml, 72 h) may have decreased apoptotic cell death. However, PBMC and THP-1 cell viability was deceased at 0.4-0.8 mg/ml C LE and 0.8 mg/ml C LE respectively, suggesting an alternative form of cell death occurred.
Increased caspase-3 and proteasome activity, as well as E3 ubiquitin-conjugating enzyme expression are associated with increased proteolysis [56]. Thus the ability of C LE to down regulate caspase activities in PBMC's and THP-1 cells may decrease proteolysis and the progression of cancer cachexia.
The cachectic syndrome is characterized by a negative energy balance due to reduced food intake and abnormal metabolism [57]. The inability to ingest/ use nutrients [5] and the negative energy balance present in cachectic patients leads to catalysis of muscle and fat stores for energy production [58]. In PBMC's, C LE decreased ATP levels, a possible consequence of the decreased cell viability. Cancer cells require high levels of ATP for cellular proliferation [59]. In THP-1 cells, C LE decreased ATP levels which may decrease THP-1 cell proliferation. However in cachexia, a decrease in ATP levels may contribute to tissue wasting.
The potent feeding stimulant neuropeptide Y (NPY) promotes food and energy intake [60]. Increased cytokine (IL-1, IL-6, TNF-α) levels may inhibit NPY signalling leading to decreased food intake and increased energy expenditure [60]. Leptin functions as a suppresser of food intake and stimulator of energy consumption [6]. Pro-inflammatory cytokines may inhibit feeding by mimicking the hypothalamic negative-feedback signalling effect of leptin [61]. Thus, the ability of C LE to decrease pro-inflammatory cytokine levels may increase food intake, decrease energy expenditure and possibly combat the negative energy balance associated with cancer cachexia.