Repeated trans-arterial treatments of LDL-DHA nanoparticles induce multiple pathways of tumor cell death in hepatocellular carcinoma bearing rats.

Introduction: Repeated hepatic arterial delivery of therapeutic agents to the liver by percutaneously implanted port-catheter systems has been widely used to treat unresectable liver cancer. This approach is applied to assess the therapeutic efficacy of repeated low-density lipoprotein-docosahexaenoic acid (LDL-DHA) nanoparticle treatments in a rat model of hepatocellular carcinoma. Methods: N1S1 hepatoma bearing rats underwent placement of a percutaneously implanted hepatic artery port-catheter system and were allocated to untreated, control LDL-triolein (LDL-TO) or LDL-DHA nanoparticle infusions groups. Treatments were performed every three days over a nine day study period. MRI was performed at baseline and throughout the study. At the end of the study tissue samples were collected for analyses. Results and Discussion: Implantation of the port catheters was successful in all rats. MRI showed that repeated infusions of LDL-DHA nanoparticles significantly impaired the growth of the rat hepatomas eventually leading to tumor regression. The tumors in the LDL-TO treated group showed delayed growth, while the untreated tumors grew steadily throughout the study. Histopathology and MRI support these findings demonstrating extensive tumor necrosis in LDL-DHA treated groups while the control groups displayed minor necrosis. Molecular and biochemical analyses also revealed that LDL-DHA treated tumors had increased levels of nuclear factor-kappa B and lipid peroxidation and depletion of glutathione peroxidase 4 relative to the control groups. Evidence of both ferroptosis and apoptosis tumor cell death was observed following LDL-DHA treatments. In conclusion repeated transarterial infusions of LDL-DHA nanoparticles provides sustained repression of tumor growth in a rat hepatoma model.

Assessing the safety of transarterial locoregional delivery of low-density lipoprotein docosahexaenoic acid nanoparticles to the rat liver.

Hepatic-arterial infusion (HAI) of low-density lipoprotein (LDL) nanoparticles reconstituted with docosahexaenoic acid (DHA) (LDL-DHA) has been shown in a rat hepatoma model to be a promising treatment for hepatocellular carcinoma. To date, little is known regarding the safety of HAI of LDL-DHA to the liver. Therefore, we aimed to investigate the deposition, metabolism and safety of HAI of LDL-DHA (2, 4 or 8 mg/kg) in the rat. Following HAI, fluorescent labeled LDL nanoparticles displayed a biexponential plasma concentration time curve as the particles were rapidly extracted by the liver. Overall, increasing doses of HAI of LDL-DHA was well tolerated in the rat. Body weight, plasma biochemistry and histology were all unremarkable and molecular markers of inflammation did not increase with treatment. Lipidomics analyses showed that LDL-DHA was preferentially oxidized to the anti-inflammatory mediator, protectin DX. We conclude that HAI of LDL-DHA nanoparticles is not only safe, but provides potential hepatoprotective benefits.

Low-density lipoprotein docosahexaenoic acid nanoparticles induce ferroptotic cell death in hepatocellular carcinoma.

Low-density lipoprotein nanoparticles reconstituted with the natural omega-3 fatty acid, docosahexaenoic acid (LDL-DHA), have been reported to selectively kill hepatoma cells and reduce the growth of orthotopic liver tumors in the rat. To date, little is known about the cell death pathways by which LDL-DHA nanoparticles kill tumor cells. Here we show that the LDL-DHA nanoparticles are cytotoxic to both rat hepatoma and human hepatocellular carcinoma (HCC) cell lines. Following LDL-DHA treatment both rat and human HCC cells experience pronounced lipid peroxidation, depletion of glutathione and inactivation of the lipid antioxidant glutathione peroxidase-4 (GPX4) prior to cell death. Inhibitor studies revealed that the treated HCC cells die independent of apoptotic, necroptotic or autophagic pathways, but require the presence of cellular iron. These hallmark features are consistent and were later confirmed to reflect ferroptosis, a novel form of nonapoptotic iron-dependent cell death. In keeping with the mechanisms of ferroptosis cell death, GPX4 was also found to be a central regulator of LDL-DHA induced tumor cell killing. We also investigated the effects of LDL-DHA treatments in mice bearing human HCC tumor xenografts. Intratumoral injections of LDL-DHA severely inhibited the growth of HCC xenografts long term. Consistent with our in vitro findings, the LDL-DHA treated HCC tumors experienced ferroptotic cell death characterized by increased levels of tissue lipid hydroperoxides and suppression of GPX4 expression. CONCLUSION: LDL-DHA induces cell death in HCC cells through the ferroptosis pathway, this represents a novel molecular mechanism of anticancer activity for LDL-DHA nanoparticles.