Kanali et al. examined the effects of 32 UA derivatives utilizing human A549 lung cancer cells and found that the analog 4-bromoanilamideursolic acid (UA-9) had the greatest anticancer effect. Accordingly, UA derivatives with electron-donating groups (UA-9) were suggested to have the most potent anticancer activity
[22]. However, all of the examined analogs had higher molecular weights and more limited polarity than UA, indicating potentially low solubility and therefore difficulty in passing through cell membranes.
Ursolic acid-triazolyl derivatives with o-bromo, o-chloro or o-methoxy substitutions on the aromatic ring showed inhibition of cell growth of cancer cells, including A549 lung cancer cells
[23]. These UA-derivative compounds displayed higher anti-cancer effects than the parent ursolic acid, suggesting these derivatives should be further examined as potential cancer treatment options
[23].
A group of ursolic acid benzylidene derivatives were created through an oxidation/condensation procedure and tested against cultured cancer cells, including A549 lung cancer cells
[24]. Each of the compounds exhibited greater cancer cell cytotoxicity than UA and less cytotoxicity to normal FR-2 lung epithelial cells. The most promising derivative created was a UA derivative with 2,5-dihydroxy substitution on the aromatic ring, named 3b. Although this compound was further tested against colon cancer cells (HCT-116) and shown to induce apoptosis
[24], studies utilizing lung cancer cells are lacking.
A series of A-ring cleaved UA derivatives were prepared and evaluated in NSCLC cells (H460, H322 and H460LKB1
+/+). UA with a cleaved A-ring and a secondary amide at C
3 (compound 17) was found to be the most active in inducing apoptosis. Treatment of lung cancer cells with this UA derivative induced apoptosis, as evidenced by the increased cleaved caspase-8 and caspase-7 levels and the decrease in Bcl-2 protein levels. Increased levels of Beclin-1 and LC3A/B-II suggested an induction of autophagy with this UA-derivative treatment. Decreases in mTOR and p62 protein levels were also observed. Based on these findings, UA derivative compound 17 may be a potential candidate for lung cancer treatment and should be further researched
[25].
Human lung cancer cell (A549 and H460) treatment with the UA derivative UA232 resulted in reduced cell proliferation and induction of apoptosis. These effects were more prominent with UA232 than treatment with the parent UA compound, whereas the cytotoxic effects on normal cells (HEK293T) were the same. UA232 treatment of lung cancer cells caused the cells to arrest in the G0/G1 phase of the cell cycle in association with downregulation of cyclin D1 and CDK4. This novel UA derivative significantly increased apoptosis and increased cleaved-PARP1 protein levels compared to standard UA treatment. There was no significant change in the levels of Bax, Bcl-2 or caspase-8, indicating that apoptosis was not induced through either the mitochondrial apoptosis pathway or the death receptor pathway. UA232 treatment increased expression of CHOP, indicating a mechanism involving the ER stress pathway. Pretreatment with 4-PBA, an ER stress inhibitor, attenuated the UA232-induced apoptosis, a further indication of the role of ER stress
[26].
Another UA derivative with functionalized aniline or amide side chains was synthesized and used to treat lung adenocarcinoma NCI-H460 cells. Compound 5Y8 had the most potent antiproliferative activity, which was significantly higher than that associated with treatment with the UA parent compound. Molecular docking studies revealed that compound 5Y8 had a key interaction with the active site of NF-kB, blocking the activity and signaling pathway, which led to apoptosis. These findings indicate that the 5Y8 UA derivative has potential as a new class of NF-kB inhibitor for the treatment of lung cancer and may contribute to overcoming chemotherapy resistance
[27].
Treatment of A549 lung cancer cells with a number of UA derivatives containing long-chain diamine moieties resulted in significant inhibition of viability, with IC
50 values in a micromolar range (5.22–8.95 μM)
[28]. The UA derivative compound 8C was the most potent (IC
50: 5.22 μM) and caused G1 phase cell cycle arrest and increased caspase 3 cleavage, an indicator of apoptosis. In addition, cancer cell migration was inhibited, suggesting that this compound may help to prevent metastasis. Treatment with UA compound 8C inhibited the NF-kB signaling pathway, as evidenced by the significantly reduced levels of phosphorylated IKKα/β and IKBα and reduced NF-kB levels. Furthermore, molecular docking studies showed a key interaction between compound 8C and the active site of NF-kB, blocking its activity
[28]. Activated NF-kB is associated with increased cancer cell proliferation and inhibition of apoptosis; therefore, NF-kB inhibitors have potential as anticancer agents. This study
[28] shows clear evidence of UA compound 8C targeting/inhibiting NF-kB activity in lung cancer cells; therefore, its anticancer potential should be further explored.
Although the parent ursolic acid compound has been shown to be an effective anticancer agent, the above-described studies have shown that derivatives of the parent UA compound have enhanced potency, indicating that modification may be an effective approach to drug development.
UA has limited bioavailability
[29][30] due, in part, to its low water solubility and high molecular weight of 456.7 g/mol. A limited number of studies have examined UA bioavailability. Mice fed a diet containing 0.05% UA for 8 weeks were euthanized, and plasma UA levels and tissue distribution were measured utilizing high-performance liquid chromatography–mass spectrometry (HPLC-MS)
[29]. Plasma UA levels of 580 ng/mL (1.26 µM) were observed, with the highest distribution in the liver (9.7 µg/g), followed by the colon (6.4 µg/g), kidney (5.9 µg/g), heart (3.9 µg/g), bladder (2.9 µg/g) and brain (1.6 µg/g). These data indicate that after oral administration of UA, significant plasma levels (micromolar) and tissue distribution can be achieved. In another study, Yang et al. prepared UA nanoparticles and measured plasma levels in rats after oral administration. One hour after rats were orally administered the parent UA or the nanoparticles at a dosage of 100 mg/kg of body weight, the resulting plasma levels were approximately 300 ng/mL (656.89 nM) and 1200 ng/mL, respectively. Plasma concentration dropped within 4 h in both groups of animals and remained at approximately 100 ng/mL (218.96 nM) for the next 12 h
[30]. This study clearly indicates that although UA nanoparticles have increased gastrointestinal absorption, the parent UA compound is sufficiently absorbed to reach significant plasma levels (nanomolar). In another study UA or UA-phospholipid complex was administered at a dose of 10 mg/kg body weight to male rats, and the same trend was observed: an increase in plasma concentration to approximately 60 ng/mL (131.38 nM) and 125 ng/mL, respectively, after one hour, which rapidly reduced to approximately 10 ng/mL (21.90 nM) by hour five and remained at that level for the remining 24 h of the experiment
[31]. However, no studies have been conducted examining UA plasma levels in humans after oral administration. In one study, subjects administered intravenous infusion of UA nanoliposomes (98 mg/m
2) had increased plasma concentration with the peak UA concentration of 3404.6 ng/mL (7454.78 nM) four hours post infusion, followed by a rapid decline between hours 4 and 6. The concentration slowly declined from post-infusion hour six (approximately 300 ng/mL (656.89 nM)) to hour sixteen (approx. 30 ng/mL (65.69 nM))
[32].
Collectively, the limited animal studies indicate that oral administration of UA can result in plasma UA levels in the nano-to-micromolar range, at concentrations close to those used in the majority of the in vitro studies showing potent anticancer effects.
Another point that deserves consideration is the role of the gut microbiome in UA-induced effects. The gut microbiota may influence UA metabolism and result in the generation of UA metabolites with potent anticancer properties. Unfortunately, the anticancer properties of UA metabolites have not been studied to date.
4. Patent Applications and Clinical Trials Related to Ursolic Acid Use
A search of patent applications (using
GooglePatents.com, accessed on 9 September 2022) revealed that in the past 10 years, 25,638 patent applications have been filed globally pertaining to UA, of which 5793 were related to UA use in cancer. In Canada, 354 applications were filed for UA patents, with 140 related to cancer. In the US, the numbers were higher, with a total of 1341 applications related to UA and 542 specifically related to UA and cancer. A search of European Union (EU) Clinical Trials Registry and the Government of Canada clinical trials registry yielded no registered trials related to ursolic acid use specifically for cancer treatment or any other use.
A search of
ClinicalTrials.gov (accessed on 9 September 2022) related to ursolic acid revealed four trials: three completed and one withdrawn. Among the completed clinical trials, one (NCT02401113) examined the effect of ursolic acid (derived from loquat extract) in preventing sarcopenia in 54 adults, although no data/results have been published (trial completed Oct 2015). Another trial examined the bioavailability of ursolic acid in 18 healthy male adult participants (NCT04421716); although the study was completed in April 2021, no results have been published. The third trial (NCT02337933, trial completed Sept 2015) examined the effects of 12-week ursolic acid administration (150 mg administered orally once a day) in 24 adult participants with metabolic syndrome
[33], with findings of reduced body weight, BMI, waist circumference and fasting blood glucose levels and improved insulin sensitivity. Approximately 50% of patients had transient remission of their metabolic syndrome. Patients with insulin resistance and metabolic syndrome are at an increased risk of developing cancer in general; although this clinical trial was not focused on cancer patients, the data are indirectly relevant to cancer. The reduced metabolic syndrome symptoms with UA use suggest improved metabolic control and potentially reduced cancer risk. One clinical trial (NCT04403568) involving the use of UA (150 mg, twice a day) for the treatment of prostate cancer was posted in May 2020. Unfortunately, this trial was withdrawn due to lack of funding.
These data clearly indicate that although there is evidence from in vitro studies and limited in vivo animal studies of the anticancer potential of ursolic acid, interest from the scientific community to perform clinical trials is limited, possibly due to a lack of strong in vivo animal studies, leading to a lack of funding.
One potential limitation of clinical translation of UA is its low bioavailability due to its low water solubility. An innovation that can combat this limitation would be to successfully encapsulate UA into micelles, nanoparticles or liposomes. These encapsulation strategies can increase the water solubility of UA, hopefully resolving the low bioavailability issue. In one preliminary clinical trial, the maximum tolerated dose (MTD), as well as the dose-limiting toxicity (DLT) of ursolic acid liposomes (11–130 mg/m
2, administered by a 4 h intravenous infusion), was investigated in a group of 63 volunteer subjects. The DLT was found to be between 74–130 mg/m
2 and mainly consisted of diarrhea and hepatotoxicity, and the MTD was determined to be 98 mg/m
2 [34]. The above study
[34] and the study by Xia et al.
[32] described in
Section 2.3, (intravenous infusion of UA nanoliposomes in humans) and the study by Yang et al.
[30] described in
Section 2.3 (oral administration of UA nanoparticles in rats) are the only studies published to date that attempted to examine dose, bioavailability and toxicity of UA nanoparticles. Furthermore, no studies have been conducted examining the effects of such UA nanoparticles against lung cancer (or any other cancer).
Although few UA derivatives have been found to be more effective than the parent compound in cell culture studies (studies presented in
Section 2.3), none of them have been tested in animal or human studies. The few studies showing chemosensitizing
[8][13][14] and radiosensitizing
[20] properties of UA, have all been conducted in cell cultures. One potential future application of UA and UA derivatives is to be used as chemo- and/or radiosensitizing agents; therefore, researchers hope that in vivo studies utilizing lung cancer animal models will be performed in the future to evaluate such a potential.
5. Conclusions
In vitro studies reported here suggest that treatment of lung cancer cells with UA reduces proliferation and viability while increasing apoptosis and autophagy. Some studies showed cell cycle arrest in the G0/G1 phase, as well as reduced cell migration. Several studies showed inhibition of NF-kB and/or mTOR pathways, which are involved in cell proliferation and survival. In most of the studies examined herein, apoptosis was confirmed through increased levels of cleaved PARP, as well as caspases 3, 7 and 9. Some studies showed an increase in the expression of the proapoptotic protein Bax and a significant decrease in the expression of the antiapoptotic protein Bcl-2, highlighting the apoptotic effects of ursolic acid on lung cancer cells.
In vivo studies revealed that treatment of lung cancer xenografted animals with UA resulted in a decrease in tumor volume and weight; however, it is not yet clear whether the mechanisms involved are the same as those reported with cell cultures.
A number of UA derivatives and analogs have been developed and tested for their effect against lung cancer, with some showing higher anticancer effects than the parent UA compound. Further studies with derivatives are needed to examine the signaling pathways involved.
Drugs currently used for the treatment of lung cancer include cisplatin, gemcitabine, docetaxel, etoposide, paclitaxel and vinorelbine. Only a few studies have compared the effects of UA to the effects of currently used lung cancer drugs. Kim et al.
[8] found that UA had similar effects as doxorubicin and veliparib in A549 lung cancer cells. Furthermore, when used in combination, UA enhanced the effect of doxorubicin and veliparib. In another study
[13], UA was reported to overcome paclitaxel resistance in A549 cells.
In vivo animal studies have shown that UA treatment of animals xenografted with lung cancer cells had a similar effect in reducing tumor volume as cyclophosphamide
[19], etoposide
[8] and doxorubicin
[8] and enhanced the effects of etoposide and doxorubicin when used in combination
[8]. Although very limited, these studies provide strong evidence of the anticancer potential of UA.
Overall, the studies summarized in the present discussion collectively show that treatment of lung cancer cells with ursolic acid considerably reduces key cancer features, including cell viability, proliferation, colony formation and migration, in addition to inducing cancer cell death. Treatment of animal models of lung cancer with UA resulted in reduced tumor volume. Future research is required to further determine the effects of UA on both cancerous and normal tissues, as well as to elucidate the cellular signaling pathways involved.
It should be noted that UA has low water solubility and limited bioavailability. Future studies should be conducted to better understand the best route of administration, pharmacokinetics, bioavailability and tumor-reducing potential of UA, its derivatives and metabolites.
Importantly, to further investigate the anticancer potential of UA in cancer patients, clinical trials are necessary.