Ricinoleo hydroxamic acid (RHA) :

Hydroxamic acids (HA) and their derivatives are weak organic acids with low toxicity, which have been widely studied for over half of a century. Several biological, biotechnological and chemical applications for hydroxamic acids and their derivatives have been reported, such as metal extraction chelators, cell-division factors, food additives, growth factors, antimicrobial agents, antimalarial drugs, tumor inhibitor drugs and enzyme inhibitors.

Hydroxamic acids (HA) is a potent anticancer moiety due to its histone deacetylases (HDAC) enzyme activity [1]. Especially, HDAC inhibitors have shown the ability to impact the level of acetylation and deacetylation of histones. Histones are core proteins of nucleosomes in chromatin with a great impact on gene expression and transcription [2]. Therefore, HDAC inhibitors can induce tumor growth inhibition, cell differentiation, and consequently programmed cell death.

Ricinoleo hydroxamic acid (RHA) is a hydroxamic acid derivative of ricinoleic acid, the main fatty acid found in castor oil. It’s mostly of interest in chemistry, biochemistry, and materials science.

It potentially has antimicrobial property and recently has been showed anticancer property against melanoma and glioblastoma.

The product is currently in the research and development (R&D) stage, and the described properties are based on early laboratory findings.

Evaluation of the cytotoxicity of the synthesized RHA using MTS cell proliferation assays revealed the anticancer activity of RHA against melanoma and glioblastoma cancer cells. The synthesized RHA showed toxicity against HDF cells also, however, HDF cells treated with 50 μg/mL RHA were able to grow up to day 3 of incubation.
This suggests that this concentration can be used to inhibit the growth of melanoma and glioblastoma cancer cells while at the same time HDF cells were able to continue their growth after treatment.

Fourier Transform Infrared (FTIR) Spectra of RHA  :

1H Nuclear Magnetic Resonance (1H NMR) spectra of RHA:

Cytotoxicity of RHA :

To assess the anticancer properties of the synthesized RHA, the cytotoxicity of various concentrations of RHA was tested against melanoma and glioblastoma cancer cell lines (Figure 1). Results showed that using as low as 50 μg/ mL RHA inhibited the growth of melanoma and glioblastoma cells completely. The average number of melanoma cells inside of wells containing 0 and 10 μg/mL RHA were in the same range at 1, 3, and 5 days post-treatment (Figure 1C). Consequently, the IC50 values obtained for the synthesized RHAwere 19.33, 34.38, and 13.22 μg/mL at 1, 3, and 5 days post-treatment of melanoma cells, respectively.

Nonetheless, IC50 values for melanoma cells were significantly lower than IC50 values for HDF cells which suggests higher toxicity of RHA against cancer cells as compared to healthy cells. Similarly, 50 μg/mL of RHA and higher concentrations (ie, 100, 500, and 1000 μg/mL) were able to inhibit the growth of glioblastoma cancer cells completely.
The average number of glioblastoma cells inside wells treated with 0 and 10 μg/mL were at the same range at 1, 3, and 5 days post-incubation. The IC50 values obtained for RHA were in the same range (ie, 33.65, 33.58, and 32.97 μg/mL at day 1, 3, and 5 post-incubation, respectively) and lower
than the IC50 values related to the HDF cells.

 Figure 1 : (A) Representative live/dead images of HDF cells 24 h post incubation. Green color represents live cells and red color represents dead cells. (B) HDF cell numbers in the presence of different concentrations of nano RHA 24 and 48 h post treatment. (C) Melanoma cell numbers in the presence of different concentrations of nano RHA after 24 and 48 h post treatment. (D) Glioblastoma cell numbers in the presence of different concentrations of nano RHA after 24 and 48 h post treatment. Data are presented as mean ± SD (n=3 and ****p < 0.0001).

Reactive Oxygen Species Assay:

To assess the mechanism behind the anticancer properties of the synthesized RHA, ROS generation was measured for various concentrations of RHA (Figure 1). RHA treatment of melanoma cells increased the production of ROS in a dosedependent manner after a concentration of 50 μg/mL.

Previously, ROS accumulation was reported for other HDAC inhibitor (HDACi) treatments as well. Although the reason behind the initially elevated ROS generation in response to HDACi remains unclear, elevated ROS generation activates different normal cell death pathways. Nonetheless, it has been shown before that cancer cells seem to have increased endogenous ROS generation in vitro and in vivo, compared to normal cells. Therefore, cancer cells are more vulnerable to further oxidative stress induced by exogenous ROS generating agents, such as RHA. This is most likely the reason why RHA treatment showed lower
toxicity against HDF cells as compared to melanoma cells after 1, 3, and 5 days post-treatment.

Figure 2 : ROS generation of melanoma cells in the presence of various concentrations of RHA at 24 h post-treatment. Data are presented as mean ± SD (n=3 and ****p < 0.0001).