HepG2 cells, Hep3B cells, Huh-7 cells, QGY-7703 cells, which are very well studied human hepatoma and hepatocellular carcinoma cell lines are used in this study. HepG2 and Hep3B were obtained from the American Type Culture Collection, Manassas, VA, USA. Huh-7 and QGY-7703 cells were a kind gift from Dr. Devanand Sarkar, Virginia Commonwealth University, Richmond, VA, USA. HepG2 cells, Huh-7 cells, QGY-7703 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum supplemented with 10% FBS and 1% penicillin/streptomycin, and Hep3B cells were grown in MEM alpha with 10% FBS, 5% Sodium Pyruvate, 5% Non-essential amino acids, and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO₂ and 18% O₂. When cells reached 80–90% confluence of growth, they were trypsinized and seeded in different culture plates or flasks based on our experimental needs.

Cell proliferation was evaluated by Water Soluble Tetrazolium-1 (WST-1) Cell Proliferation Assay System (Roche Diagnostics, Rotkreuz, Switzerland). HCC cells were seeded in 96-well plates at 5 × 10³ cells per well and treated with Withaferin A (5 µM) at 37°C under 5% CO₂ for 24 h. At the end of the 24 h period, 10 µl premixed WST-1 reagent was added to each well, and the plates were incubated further for 2 h at 37°C under 5% CO₂. Thereafter, absorbance was measured at 450 nm using a Turner-Biosystems microplate reader.

Colony formation assay was carried out using Huh-7 and QGY-7703 cells. The cells were seeded in 6 cm dishes at a density of 500 cells per plate and treated with Withaferin A (5 µM) and cultured for about 14–16 days until the colonies were visible. The cells were fixed in formaldehyde for 20 mins and washed with running tap water and stained with 10% Giemsa (Sigma-Aldrich, St. Louis, MO, USA). After rinsing and washing with running tap water, the plates were air dried, visualized under the microscope, and photographed. The images were analyzed using NIH ImageJ software and colonies counted and numbers showed in the bar graph.

Wound healing assay was carried out using Huh-7 and QGY-7703 cells (2 × 10⁵ cells/3 ml). The cells were seeded in a six-well plate and incubated at 37°C until cells were 90% confluent. A scratch was made using a 100–200 ml pipette tip, followed by washing with PBS to remove cell debris, and then treated with 5 μM Withaferin A in a complete medium. After 24 h of incubation, the cells were observed under a light microscope and randomly chosen fields were photographed at 20× objective. The percentage of Huh-7 and QGY-7703 cells migrated into the scratched area was calculated using ImageJ software.

Transwell Invasion assay was conducted using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Franklin Lakes, NJ, USA) as suggested in the manufacturer’s instructions. Pre warmed serum free media was added to the bottom side of the transwell as well as the upper chamber above the matrigel for 2 h at room temperature for rehydration. Huh-7 and QGY-7703 cells (5 × 10⁴ cells) were seeded in the upper chamber in serum free medium (with or without 5µM Withaferin A) while the wells of the lower chamber were filled with complete medium (5% FBS). After 22 h of incubation at 37°C and 5% CO_2, the cells on the upper surface of the transwell filters were removed by gentle wiping with a cotton swab and the cells attached on the lower surface of the filters were fixed and stained with Diff-Quick stain (IMEB Inc., San Marcos, CA, USA). After staining the invaded cells on the transwell filter were photographed using a microscope and invasion was determined by counting the cells using ImageJ software (6).

Anchorage-independent growth ability of HCC cells was measured by conducting soft agar colony formation assay using highly aggressive QGY-7703 cells. These cells were pretreated with vehicle control and Withaferin A for 4 h and cells were trypsinized, counted, and seeded at 10⁵ cells/plate in 6 cm dishes with culture media containing 0.4% noble agar (Sigma-Aldrich, St. Louis, MO, USA) over a 0.8% agar base layer at 37°C with 5% CO₂ for 15 days. The colonies formed were counted manually under the microscope and photographed.

Human Angiogenesis and Cytokine Arrays were carried out to measure the secreted angiogenic and cytokine markers. The QGY-7703 cells were cultured up to 70% confluence and Withaferin A was treated for 24 h and the media was changed to serum free media for further 24 h. Supernatants of cells cultured in serum free media (conditioned media) were collected, centrifuged, cell debris was separated, and the only supernatant was used to check the expression and secretion of angiogenesis associated growth factors, cytokines, and other related molecules using commercially available human angiogenesis antibody array and Human Cytokine Array kit following the manufacturer’s instructions sheets (R&D Systems, Minneapolis, MN, USA).

Total RNA was extracted from HepG2 cells treated with or without Withaferin A using TRIzol reagent (Thermos Scientific, Waltham, MA, USA). The experimental procedure was followed as described previously (6) and the primer sequences for the selected and validated LXR-α target genes are given in Table 1 .

All the data are presented as means ± SEM (n = 3). Statistical significance was analyzed using a two-tailed unpaired Student’s t-test. GraphPad Prism software (version 6) was used for all statistical analyses and p values <0.05 were considered significant.

In this study, we explored the therapeutic potential of Withaferin A on proliferation, migration, and invasion of HCC cells. HCC cells (Hep3B, HepG2, Huh-7, and QGY-7703) were treated with various doses (1, 5, and 10 µM) of Withaferin A for 24 h. The results of the WST-1 cell proliferation assay conducted at the end of the treatment period, showed that Withaferin A significantly inhibited the proliferation of HCC cells ( Figure 1A ) and the images were photographed under the microscope after the treatment of 5 µM Withaferin A to these cells ( Figure 1B ). Further, we validated the effect of Withaferin A on the colony formation ability of these cells and the results showed that more than 50% inhibition of colony formation was observed in Withaferin A treated cells compared to control cells. Colony formation assay ( Figure 1D ) and Soft agar colony formation assay ( Figures 1C, E ). Next, we determined the effects of Withaferin A (2.5 µM) on migration and invasion of QGY-7703 and Huh-7 cells by employing scratch wound-healing assay and transwell invasion assay. As shown, both the assays demonstrated that Withaferin A attenuated the migration ( Figures 2A–C ) and invasion ( Figure 2D ) of QGY-7703 and Huh-7 cells.

Here, we evaluated the effect of Withaferin A on the secretion of various angiogenesis markers and cytokines by QGY-7703 cells. Recently, few studies have shown that Withaferin A has LXR-α agonist property and it acts as a specific ligand for LXR-α (16–19). However, the significance of this property of Withaferin A and its molecular action is not studied in cancer cells. Withaferin A is also known for its anti-inflammatory properties via inhibiting the NF-κB transcription factor (20). LXR-α, a nuclear receptor family member is known to play a pivotal role in the various biological process which includes inflammation, cholesterol homeostasis, lipogenesis, cellular reprogramming, and decisions (16). Therefore, we focused our study on LXR-α/NF-κB signaling pathway, and the data supported our hypothesis. Withaferin A (2.5 µM) treatment decreased the secretion of various angiogenesis-related markers, growth factors, and cytokines (Serpin F1(PEDF), uPA, PDGF-AA, Angiogenin, Endothelin-1, Macrophage migration inhibitory factor (MIF), PAI-1, MCP1, ICAM-1 in QGY-7703 cells ( Figures 3A, B ). These factors are very well known for their pivotal role in proliferation, migration, invasion, angiogenesis, inflammation, and metastasis (21–23). It is also a known fact that NF-κB is a master regulator of various inflammatory signaling pathways (24). All these factors are directly or indirectly regulated by both NF-κB and LXR-α (25, 26). LXR-α is a negative regulator of NF-κB signaling and in this study activation of LXR-α by Withaferin A may downregulate the secretion of all these molecules via suppressing NF-κB activity.

Further, to confirm the agonistic role of Withaferin A we thought of validating some of the LXR-α target genes in HCC cells. Therefore, we treated HepG2 cells with Withaferin A (2.5 µM) for 4 h and isolated total RNA from these cells, and measured the expression of ATP-binding cassette sub-family A member 1 (ABCA1), ATP-binding cassette sub-family G member 1(ABCG1), and Apolipoprotein E(ApoE). These three genes are commonly known LXR-α target genes and were found to be significantly increased in Withaferin A treated cells in comparison with vehicle controls cells ( Figure 4 ).