Investigation of Self-Assembled Nanostructured Protein-Based Therapeutic Approaches in Breast Cancer

Development of various therapeutic approaches for the treatment of breast cancer is on the rise. Among them, chemotherapy has been widely practised for the treatment of breast cancer. However, the approach exposes the patient to various kinds of side effects caused by the drugs. Although gene therapy finds a decent approach but it is expensive and associated with delivery problems. In order to avoid the side effects of chemotherapeutic drugs and achieve a cheap and toxicity free therapeutics, the focus has been gradually shifting towards biomolecule-based therapeutics like gene, peptide and proteins as an alternate therapeutic strategy. In our present investigation, we have developed protein-based various therapeutic strategies in breast cancer which includes two proteins, bovine α-lactalbumin (BLA) and hen egg white lysozyme (LYZ). Both of them are already reported to have antitumor and anticancer activity. However no detail investigation was performed in breast cancer. Moreover, it was also reported for BLA that the specific structural state is responsible for its antitumor or anticancer activity. Here both the proteins were transformed into their self-assembled nanostructure state using simple desolvation technique through the chemical crosslinking process. Both the nanoassembly were characterized in details and their anti-proliferative activity in breast cancer cells was evaluated. The nanostructures were characterized using FESEM (Field Emission Scanning Electron Microscopy), AFM (Atomic Force Microscopy) and DLS (Dynamic Light Scattering) analysis for size and shape, FTIR (Fourier Transform Infrared Spectroscopy for bonds involved in nanoassembly), Circular dichroism and Fluorescence spectroscopy and conformational change of proteins. Microscopy and DLS analysis confirmed their average size as 300 nm. The stability of both the self-assembled nanostructures of lysozyme (snLYZ) and α-lactalbumin (snBLA) were examined against thermal, pH and protease stress and found to be highly stable. Moreover, both of them demonstrated excellent hemocompatibility in human erythrocytes as well as cytocompatibility in normal human cells like HaCaT and mouse cells like 3T3. When the nanoassemblies were applied to breast cancer cells MCF-7 and MDA-MB231, they demonstrated high ( > 90% ) cell death at 24 h and almost complete cell death after 48 h of administration. The microscopic images confirmed that the internalization of protein assemblies by MCF-7 cancer cells was the major cause of such cell death. Further, the investigation also confirmed that such death was mediated by reactive oxygen species (ROS) generation.The microscopic image and western blotting results confirmed that apoptosis had no role in cell death mechanism. The cytotoxic behaviour of snBLA was compared with a well reported anti-cancer agent BAMLET (bovine α-lactalbumin made lethal to tumor cells) in four different cancer cell lines including two breast cancer cell lines MCF-7 and MDA-MB-231. Our synthesized snBLA demonstrated better cell killing potential than BAMLET with much higher hemocompatibility. Both the nanoassembly demonstrated high Tamoxifen loading (124%) through chemical crosslinking process and release of drug efficiently in a pH-responsive manner. Furthermore the drug release process was completely regulated by conformational change of the protein in nanoassembly. In our next step we attempted to develop Graphene oxide nanosheets (GOns) and Zinc oxide nanoparticle (ZnONP) based therapeutic strategy in breast cancer to compare with our protein-based therapeutic approach. Graphene oxide nanosheets (GOns) and Zinc oxide nanoparticles (ZnONP) are being developed as drug carriers, however, such an approach was plagued with problems such as hemotoxicity and cellular biocompatibility. Hence, we made an attempt to develop a therapeutic strategy based on ZnONP and GOns by functionalizing them with BLA protein. The surface functionalization with BLA improved their hemocompatibility in human erythrocytes and cytocompatibility in normal human cells. They also demonstrated high anti-proliferative activity in breast cancer cells via the ROS-based mechanism of cell death. However, considering the consequence like protein degradation in cells and subsequent exposure of GOns and ZnONP to cells may lead to their accumulation in normal cells and tissues that might limit their use as a rational approach. Therefore, in our present study, we concluded that our developed stable self-assembled nanostructured lysozyme and α-lactalbumin can be used as a rational therapeutic strategy in breast cancer

Phytochemicals of “Magahi Pan” (Piper betle L. var. magahi) as Potential H+/K+-ATPase Inhibitors: In-Silico Study and ADME Profile

Background and objectives:  in India, peptic ulcer is most prevalent gastrointestinal disease. Historically Piper betle has been used to treat stomach problems. In order to identify the phytochemicals present in Piper betle. L. var magahi LC/MS spectroscopic analysis was performed, following which, potential phytomolecules with H+/K+-ATPase inhibitory activity were chosen using in-silico evaluation. Methods: Phytochemicals in ‘Magahi pan” were investigated and potential H+/K+-ATPase inhibitor phytochemicals that were screened through in-silico analysis and ADME profile of selected phytochemicals were evaluated. Phytochemical characterization was done with the help of LC/MS followed by molecular docking against enzyme H+/K+-ATPase (PDBID:5YLV) using Autodock4.2 and Swiss ADME. The binding affinity, free energy, physicochemical property, saturation of carbon atoms, number of hydrogen bond acceptors-donors, molar refractivity, lipophilicity, water solubility, and drug likeliness property were evaluated in-silico for their predicted bioactivity against H+/K+-ATPase. Results: A total of 67 phytoconstituents were identified through LC/MS positive and negative ionization mode spectral analysis and six were selected on the basis of binding energy. Molecular docking results revealed that the isolated compounds interacted with target protein H+/K+-ATPase with minimum binding energy ranging from (1) netilmicin (-9.29 kcal/mol); (2) benztropine (-9.07 kcal/mol); (3) 5,6,7,3’,4’ pentahydroxyisoflavone (-8.45 kcal/mol); (4) 2-O-acetylpseudolycorine (-8.02 kcal/mol);  (5) R-95913 (-7.73 kcal/mol) and (6) luteolin (-6.93 kcal/mol), respectively. Conclusion: The ADME profile analysis and docking studies revealed 5,6,7,3’,4’ pentahydroxy-isoflavone and luteolin as potential molecules for inhibiting H+/K+-ATPase

Stable Self-Assembly of Bovine α‑Lactalbumin Exhibits Target-Specific Antiproliferative Activity in Multiple Cancer Cells

Self-assembly of a protein is a natural phenomenon; however, the process can be performed under a suitable condition in vitro. Since proteins are nontoxic, biodegradable, and biocompatible in nature, they are used in various industrial applications such as biocatalyst, therapeutic agent, and drug carriers. Moreover, their flexible structural state and specific activity are being used as sensors and immensely attract many new applications. However, the inherent potential of protein self-assembly for various applications is yet to be explored in detail. In this study, spherical self-assembly of bovine α-lactalbumin (nsBLA) was synthesized using an optimized ethanol-mediated desolvation process with an average diameter of approximately 300 nm. The self-assembly was found to be highly stable against thermal, pH, and proteases stress. When nsBLA was administered in various cancer cells, it demonstrated high cytotoxicity in three different cancer cells via reactive oxygen species (ROS) generation, whereas it exhibited negligible toxicity in normal human and murine cells. When nsBLA was conjugated with folic acid, it improved the cytotoxicity and perhaps mediated through enhanced cellular uptake in cancer cells through binding with folate receptors. Further, experimental results confirmed that the cancer cell death induced by nsBLA was not caused by apoptosis but a necrotic-like death mechanism. When compared with a well-known protein-based anticancer agent BAMLET (bovine α-lactalbumin made lethal against tumor cell), the self-assembled BLA clearly exhibited higher cytotoxicity to cancer cells than BAMLET. While BAMLET exhibits poor biocompatibility, our nsBLA demonstrated excellent biocompatibility to normal cells. Therefore, in this study, we prepared self-assembled α-lactalbumin that exhibits strong inherent antiproliferative potential in multiple cancer cells which can be used for efficient therapeutic approach in cancer

Lapachol inhibits glycolysis in cancer cells by targeting pyruvate kinase M2.

Reliance on aerobic glycolysis is one of the hallmarks of cancer. Although pyruvate kinase M2 (PKM2) is a key mediator of glycolysis in cancer cells, lack of selective agents that target PKM2 remains a challenge in exploiting metabolic pathways for cancer therapy. We report that unlike its structural analog shikonin, a known inhibitor of PKM2, lapachol failed to induce non-apoptotic cell death ferroxitosis in hypoxia. However, melanoma cells treated with lapachol showed a dose-dependent inhibition of glycolysis and a corresponding increase in oxygen consumption. Accordingly, in silico studies revealed a high affinity-binding pocket for lapachol on PKM2 structure. Lapachol inhibited PKM2 activity of purified enzyme as well as in melanoma cell extracts. Blockade of glycolysis by lapachol in melanoma cells led to decreased ATP levels and inhibition of cell proliferation. Furthermore, perturbation of glycolysis in melanoma cells with lapachol sensitized cells to mitochondrial protonophore and promoted apoptosis. These results present lapachol as an inhibitor of PKM2 to interrogate metabolic plasticity in tumor cells

Lapachol inhibits melanoma cell proliferation.

<p>(A) Cell proliferation was determined for MEL 526 and MEL103 melanoma cell lines and plot show the rate of proliferation in the presence of increasing concentrations of lapachol (5 μM, 10 μM, 20 μM, 40 μM) (n = 3/condition)****P<0.0001 (B) Cellular ATP levels were measured in MEL 526 and MEL103 melanoma cell lines in the presence of increasing concentrations of lapachol (5 μM, 10 μM, 20 μM, 40 μM) (n = 3/condition)****P<0.0001.</p

In silico studies of lapachol-PKM2 interactions.

<p>(A) The structure of human muscle Pyruvate kinase M2 [1ZJH—Receptor] docked to shikonin (ligand) using Autodock 4.2 software and ligand receptor interaction diagram generated using Discovery studio 2.5. The predicted free energy of binding for shikonin -7.98 kcal/mol and predicted inhibition constant was 1.42 μM. Inset shows the site pocket with surface charge distribution on donor and acceptor atoms. (B) The structure of human Pyruvate kinase M2 docked to lapachol and ligand receptor interaction diagram generated using Discovery studio 2.5. Inset shows the site pocket with surface charge distribution on donor and acceptor atoms. (C) The 17 amino acid residues of human PKM2 protein predicted to interact with the functional groups in shikonin and the type of interaction Van der waals (pale green), H-bond (bright green), Pi-Sigma (purple) and Pi-Alkyl (pink) are highlighted. (D) The 20 amino acid residues of human PKM2 protein predicted to interact with the functional groups in lapachol and the type interaction are highlighted.</p

Lapachol sensitizes melanoma cells to apoptosis.

<p>(A) Annexin 7AAD-PE analysis of apoptosis carried our in MEL103 cell line. Treatment with DMSO (Vehicle) or 2, 4-di nitro phenol (DNP 10 μM) alone showed low percentage of annexin positive cells 7.9% and 8.2% respectively. Lapachol (20 μM) treatment increased annexin positive cells to 13% but the combined treatment of lapachol (20 μM) and DNP (10 μM) further increased the percentage of annexin positive cells to 20%. (B) Loss of mitochondrial membrane potential was analyzed by TMRM dye in MEL 103 melanoma cell line after treatment with DMSO (Vehicle), lapachol (20 μM), DNP (10 μM) and lapachol (20 μM) +DNP (10 μM). Mitochondrial membrane integrity in vehicle, lapachol (20 μM) and DNP (10 μM) treated cells and loss of mitochondrial membrane potential is revealed in cells treated with a combination of lapachol (20 μM) and DNP (10 μM) as reflected by the loss of TMRM fluorescence intensity.</p

Lapachol inhibits PKM2 enzyme activity.

<p>(A) Recombinant human PKM1 enzyme was isolated and in vitro PKM1 enzyme activity was measured using a continuous assay coupled to lactate dehydrogenase. Different concentrations of lapachol (vehicle, 5 μM and 10 μM) did not show an inhibition in the activity of recombinant PKM1 invitro. (B) lapachol inhibited the activity of recombinant PKM2 in a dose dependent manner (n = 3/condition) **** P<0.0001 (C) Lapachol inhibited the pyruvate kinase activity invivo in the melanoma cell line MEL103 in a dose dependent manner (n = 3/condition)****P<0.0001.</p

Shikonin but not lapachol promotes ferroxitosis in hypoxia.

<p>(A) Chemical structures of naphthoquinone analogs, shikonin [5,8-dihydxroxy-2-(1-hydroxy-4methyl-3-pentenyl)-1,4-naphthoquinone], menadione [2-methyl-1,4-naphthoquinone] and lapachol [2-Hydroxy-3-(-3- methyl-2-butenyl-)-1,4-naphthoquinone]. (B) MEL103 (NRAS^Q61L), MEL697 (NRAS^Q61L BRAF^V600E) and MEL526 (BRAF^V600E) were treated with 20μM menadione, 5–20 μM shikonin, or 5–20 μM lapachol for 12 hours in a hypoxic chamber. Viability was determined in a resazurin-based assay. (n = 3/condition) **** P<0.0001. (C) Table depicting the ability of menadione, shikonin and lapachol to induce ferroxitosis under normoxic and hypoxic conditions (“+”—induces ferroxitosis, “_”- does not induce ferroxitosis).</p