[19]. The model created by this research group provides preliminary evidence of UA acting against cigarette-smoke-induced changes in lung cells. However, this is an in vitro model, and further work utilizing animal models is required to determine whether UA protects against cigarette-smoke-induced damage. Strong evidence of a protective role of UA in animal models would provide a justification for clinical trials.
A549 cells treated with UA showed reduced proliferation, as well as reduced adhesion, wound healing and transwell migration, indicating inhibition of metastasis. Western blot analysis revealed an upregulation of E-cadherin and a downregulation of N-cadherin and vimentin, indicating an inhibition of epithelial–mesenchymal transition (EMT). Furthermore, decreased expression of astrocyte-elevated gene-1 (AEG-1) correlated with repression of NF-kB signaling was observed in cells treated with UA
[6].
Treatment of A549 and H460 human lung cancer cells with UA significantly decreased cell viability and proliferation. UA increased chromatin condensation and induced apoptosis. Flow cytometry revealed an increase in cell cycle arrest in the G0/G1 phase. Western blot analysis revealed an increase in cleaved caspases 3 and 9, increased phosphorylation of the proapoptotic protein Bax and a decrease in phosphorylated Bcl-2, an anti-apoptotic protein, suggesting that UA induces apoptosis in these cells via a caspase-dependent pathway. Treatment with UA increased phosphorylation/activation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation/activation of mTOR and p70S6K proteins, which are involved in protein synthesis and cell growth. In addition, the levels and activity of the lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) were decreased due to increased AMPK activation
[7]. These data clearly show that UA is a strong activator of the AMPK energy sensor. UA-induced AMPK activation was abolished by an siRNA approach, which downregulated LKB1. Activated AMPK leads to downstream inhibition of mTOR and p70S6K, resulting in inhibition of protein synthesis and, ultimately, inhibition of cell proliferation. The inhibition of lipogenesis is also the result of activation of AMPK, resulting in limited availability of lipids, which are important components for cell proliferation. Although the study did not provide any evidence, it is possible that the activation of AMPK is also responsible for the induction of apoptosis. An approach to knockout/downregulate AMPK would have provided stronger evidence of the role of AMPK in the UA-induced effects.
Treatment of A549 human lung cancer cells with ursolic acid (0–50 µM) resulted in reduced cell viability and increased DNA damage associated with inhibition of vaccinia-related kinase 1 (VRK1) autophosphorylation and reduced phosphorylation of its downstream substrates, CREB and histone H3. This UA treatment also decreased cyclin D1 mRNA levels, a downstream effector of CREB and histone H3. Nuclear magnetic resonance (NMR) titration experiments and in silico modeling showed that UA is able to bind directly to VRK1, causing inhibition of kinase activity. A combination of UA with doxorubicin resulted in greater reduction in cell viability compared to either compound alone, indicating a synergic effect. Furthermore, when UA was combined with the PARP-1 inhibitor veliparib, a significant reduction in cell viability was observed compared to UA alone, indicating that UA exerts synergistic effects when combined with drugs that target DNA damage repair genes
[8]. The finding reported in this study, that UA binds directly to the catalytic domain of VRK1 inhibiting its kinase activity, is based on in silico modeling. Further work is required to confirm that UA binds to VRK1 in cells, and the use of VRK1 overexpression/knockdown (siRNA) approaches can provide evidence of the role of VRK1 in UA-induced apoptosis. The finding that UA enhanced the effect of doxorubicin and veliparib is significant, suggesting that UA has the potential to be used as an adjuvant chemotherapy agent against lung cancer.
Wu et al. reported significantly reduced cell viability and induction of apoptosis in NSCLC (H1299, H1650, A549, PC9 and H1975) cells treated with UA (5–80 µM). UA treatment increased phosphorylation/activation of stress-activated protein kinases/Jun amino-terminal kinases (SAPK/JNK) while simultaneously decreasing the protein levels of SP1, DNMT1 and EZH2. Detection of caspase 3/7 activity was increased by UA treatment, as observed with a Caspase 3/7 assay kit. Use of the SAPK/JNK inhibitor SP600125 abolished the effects of UA on SP1, DNMT1 and EZH2, confirming the involvement of this signaling pathway. Exogenous expression of SP1 or DNMT1 also abolished the UA-induced effects, providing strong evidence of their involvement. Based on these data, the authors concluded that UA inhibits NSCLC cell growth via SAPK/JNK-mediated inhibition of SP1, DNMT1 and EZH2
[9].
Treatment of mesothelioma cells (H28, H2452 and MSTO-211H) with 10–40 µM UA inhibited proliferation and colony formation in a dose-dependent manner. Cell-cycle analysis showed an accumulation in the sub-G1 phase compared to untreated controls, indicating induction of apoptosis. Western blot analysis showed reduced total PARP and reduced caspase 3, both of which are apoptosis-related proteins. UA treatment attenuated the levels of EMT-related proteins; E-cadherin expression was increased, whereas levels of vimentin, N-cadherin, twist, B-catenin and pGSK3a/b were decreased. The expression of the survival genes cyclin D1, p-AKT and NF-kB were also reduced with UA treatment. MicroRNA array and qRT-PCR results indicated that let7b was upregulated in H2452 and H28 cells treated with UA. Overall, the data reported in this study suggest that UA treatment can induce apoptosis in lung cancer cells through inhibition of EMT and increased expression of let7b
[10]. Downregulation of let7b would have provided stronger evidence of its role in the UA-induced effects.
Song et al. (2017) examined the ability of UA to increase the radiosensitivity of H1299 NSCLC cells
[20]. They established radioresistant H1299 NSCLC cells by expressing a mutant hypoxia inducible factor-1α (HIF-1α) and found that treatment with UA increased their radiosensitivity (as evidenced by reduced cell survival), and this effect was associated with reduced endogenous GSH and HIF-1α levels
[20]. Although these findings suggest that UA has the potential to radiosensitize lung cancer cells in vitro, further studies are required to examine whether UA causes radiosensitization in animals xenografted with NSCLC cells. Once strong evidence is reported from in vivo animal studies, clinical studies will be required to examine whether UA causes radiosensitization in NSCL patients.
Treatment of A549 cells with UA (10–100 µM) for 24 h inhibited cell proliferation and induced apoptosis. UA decreased the number of cells in the G0/G1 phase in a dose-dependent manner and increased the number of cells in the S and G2/M phases, suggesting UA-induced cell cycle arrest in the S and G2/M phase. This UA treatment decreased protein expression of Bcl-2 and total PARP, increased levels of cleaved-PARP and increased markers for autophagy, including the LC3-II/LC3-I ratio and p62 protein expression
[11].
Human lung cancer A549 cells treated with UA (10, 20 and 40 µg/mL) for 24 or 48 h showed reduced cell viability, as well as increased autophagy and mitophagy. Treatment with UA increased the levels of autophagy-associated protein LC3-II and increased the LC3-II/LC-I ratio. In addition, the mitochondria of cells treated with UA appeared to be fragmented, and the levels of p62 protein, which is required for mitophagy, were increased and colocalized with fragmented mitochondria. Furthermore, treatment with UA increased ROS production, increased Nrf2 and PTEN-induced kinase 1 (PINK1) protein levels and reduced phosphorylated Akt and mTOR levels
[12]. The data clearly show inhibition of Akt and mTOR as a result of UA treatment and not activation, as stated in the abstract (possible typographical error). The data from the two above-mentioned studies
[11][12] suggest induction of autophagy and mitophagy by UA treatment; however, whether autophagy and mitophagy are induced in lung tumors in vivo is not known.
Ursolic acid treatment of NSCLC H1975 cells with an EGFR T790M mutation, a major cause of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI/erlotinib) resistance, resulted in inhibition of proliferation and motility, as well as induction of apoptosis. UA treatment resulted in inhibition of CT45A2 expression via β-catenin/TCF4 inhibition. These data show that UA may be a potential treatment for NSCLC with the EGFRl858R/T790M mutation
[14]. This study included examination of the effects of UA in animals xenografted with lung cancer cells expressing the EGFR T790M mutation; the findings are reported below (see in vivo section).
Chen et al. constructed a paclitaxel resistance cell line, A549-PR, with increased expression of stemness markers CD133, Oct-4, Notch3, Nanog and Sox2. Treating these chemotherapy-resistant cells with UA resulted in decreased expression of these marker proteins, reduced aldehyde dehydrogenase 1 (ALDH1) activity and, importantly, increased sensitivity to paclitaxel. Treatment of A549-PR cells with UA resulted in reduced miR-149-5/MyD88 signaling. The UA-mediated inhibition of chemoresistance and stemness was partially reversed by overexpression of miR-149 and knockdown of MyD88
[13]. Paclitaxel is a chemotherapy agent commonly used in the treatment of lung cancer. Unfortunately, many patients treated with paclitaxel develop resistance; therefore, the identification of methods to overcome such resistance is highly desirable, as it will improve patient survival. The findings of this study are significant; however, they are derived from in vitro studies only. Further in vivo animal studies and human clinical trials are required to confirm whether UA has the ability to overcome paclitaxel resistance.
Human NSCLC cells (H1975) treated with UA exhibited inhibited mesenchymal-like responses, such as migration, invasion and matrix metallopeptidase (MMP) 2 and 9 activity
[15]. Furthermore, treatment with UA attenuated the transforming growth factor-β1 (TGF-β1)-induced decrease in E-cadherin and increased N-cadherin levels. TGF-β1-induced αVβ5 integrin levels and cell migration and invasion (using scratch and transwell invasion assays) were attenuated by UA treatment. The use of a pharmacological inhibitor of integrin (SB273005), similarly to UA treatment, reduced TGF-β1-stimulated cell migration and invasion, whereas combined treatment with UA and the inhibitor did not have any synergic effect. Although these data indicate that UA may target/reduce αVβ5 levels, leading to reduced epithelial-to-mesenchymal transition in NSCLC
[15], more robust evidence is required to confirm this phenomenon. The use of inhibitors in general may lead to inhibition of other off-target molecules, and the specificity of SB273005 against αVβ5 versus other integrins is not clear. In addition, it is not known whether UA can bind to αVβ5. Further studies are required to answer these questions.
Human lung cancer cells (NCI-H292) treated with UA showed significantly reduced viability and increased apoptosis, as evidenced by increased levels of cleaved PARP and increased cytochrome C levels. Western blotting analysis revealed increased levels of endonuclease G (Endo G) and apoptosis inducing factor (AIF), whereas levels of the antiapoptotic proteins B-cell lymphoma 2 (BCL2) and BH3 interacting domain death agonist (BID) were reduced with UA treatment. In these cells, UA increased intracellular calcium levels by causing a time-dependent release and reduced mitochondrial membrane potential without affecting ROS levels. Taken together, these results suggest that UA treatment can induce apoptosis through AIF and Endo G release via a mitochondria-dependent pathway
[16].
A variety of lung cancer cells (NSCLC: A549, H1975, H1299, H460 and H520; SCLC: H82 and H446; murine Lewis lung carcinoma cell line, LLC), when treated with UA, exhibited significantly reduced cell proliferation, whereas apoptosis was induced, as evidenced by an annexin V-FITC/PI double staining assay. UA treatment increased levels of cleaved PARP and decreased levels of Bcl-2, both indicators of apoptosis. In addition, treatment of H460, H1975 and A549 lung cancer cells with UA induced autophagy, as revealed by the elevation in the protein levels of the autophagy marker LC-3-II. Furthermore, UA treatment decreased phosphorylation/activation of Akt and its downstream targets, p70S6K and 4EBP1. Inhibition of autophagy, using siRNA for autophagy-related gene 5 (ATG5) or chloroquine, enhanced the effects of UA (inhibition of proliferation and induction of apoptosis), suggesting that the autophagy observed following UA treatment acts as a pro-survival mechanism in these cells
[17].
Human A549 and H460 lung cancer cells treated with UA exhibited reduced proliferation, as well as reduced CDK4, cyclin D1 and cyclin E protein and mRNA levels, whereas the levels of p21 and p27 tumor suppressor proteins were increased. In addition, treatment with UA resulted in decreased VEGFR and pSTAT3 levels, suggesting inhibition of angiogenesis. Decreases in cell invasion and wound healing/migration were observed, suggesting antimetastatic activity of UA in NSCLC cells. Cancer stem cell (CSC) proliferation was inhibited with UA treatment, as evidenced by a significant reduction in the levels of CSC markers (NANOG, POU5F1 and SOX2). Molecular binding studies showed a high-affinity interaction between UA and EGFR, suggesting that UA interferes with EGFR signaling. This was further tested by treating cells with human recombinant EGF combined with 20 µM of UA. In the presence of UA, the EGF-mediated EGFR phosphorylation was reduced, and the downstream targets of the EGFR pathway, JAK2 and STAT3, showed reduced phosphorylation, whereas the total protein levels remained unchanged. Chromatin DNA extracted from NSCLC cells that had been treated with UA exhibited a significant reduction in the binding of STAT3 to promoters MMP2 and PD-L1. Based on these results, the authors concluded that UA has anticancer activity dependent on STAT3 signaling, that STAT3 acts as a bridge between EGFR and PD-L1 and that UA may be a drug candidate for PD-L1-based targeted cancer therapies
[18]. Although the authors suggested that UA acts as an inhibitor of EGFR, further studies are required to confirm this hypothesis. Future in vitro studies utilizing recombinant EGFR and UA can clarify this issue.
According to the results of the studies presented above, it is evident that the effects of UA were examined in a variety of lung cancer cells representing distinct subtypes of the disease. Small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) adenocarcinoma, squamous cell carcinoma and large cell carcinoma cell lines were utilized. The effective concentration of UA appears to be in the range of 20–50 µM in the majority of the studies reviewed herein. Unfortunately, no studies have been conducted that examine and directly compare the UA concentration required for half-maximum inhibition (IC
50 values) of proliferation of different lung cancer cell lines.
Overall, the in vitro studies presented above and in
Table 1 show that treatment of lung cancer cells with UA results in inhibition of proliferation, induction of apoptosis and autophagy through inhibition of Akt, NF-kB and mTOR; increased LC3-II/LC-3-I ratio; and increased cleavage of caspase 3/7 and PARP (
Figure 1).