Ananda Chakrabarty, PhDDistinguished University Professor
PhD, University of Calcutta, India
My laboratory studies a family of proteins known as cupredoxins and cytochromes that are involved in bacterial electron transport. While these proteins have been studied extensively for their electron transfer properties, they were not known to have any other function. Over the last 8 years, we have shown that some of these proteins enter mammalian cells, particularly human cancer cells, and induce either growth arrest by inhibiting cell cycle progression at the G1 phase, or apoptosis in cancer cells. With azurin, a cupredoxin from the pathogen Pseudomonas aeruginosa believed to be involved in electron transfer during denitrification, we have shown that azurin enters into human melanoma and breast cancer cells and kills them by inducing apoptosis through release of mitochondrial cytochrome c to the cytosol which leads to caspase-9 activation. Most interestingly, azurin not only kills human cancer cells in vitro by stabilizing the tumor suppressor protein p53, but it also allows 59 to 85% in vivo regression of human melanoma and breast cancers, when these tumors are xenografted in immunodeficient nude mice. Another cupredoxin, rusticyanin, produced by a nonpathogenic bacterium Acidithiobacillus ferrooxidans, which is normally involved in electron transfer during oxidation of Fe2+ to Fe3+, can also enter human cancer cells and induces apoptosis by generating reactive oxygen species (ROS) and activating caspase-8 but not caspase-9. Thus while both these cupredoxins kill human cancer cells, their modes of action are different. Azurin has also been shown to enter cancer cells preferentially and inhibit angiogenesis as well as cell signaling by complexing with a receptor tyrosine kinase. Thus azurin inhibits cancer growth by interfering in multiple pathways in addition to inducing programmed cell death. Because such proteins and their truncated derivatives can potentially be used as anticancer agents in the treatment of human cancer, we have filed appropriate patent applications and have raised enough funding from private investors for carrying out human clinical trials.
We have also recently demonstrated that an azurin-like protein called Laz from Neisseria enters in brain tumor glioblastoma cells and kills them, while azurin from P. aeruginosa has much less effect. Laz is also very efficient, as compared to azurin, to bind proteins on the surface of the malarial parasite Plasmodium falciparum,or the parasite Toxoplasma gondi and on the envelope of the AIDS virus HIV-1. This allows Laz and azurin to significantly inhibit the entry to and growth of HIV-1 and P. falciparum or Toxoplasma gondi in the host cells. Most modern drugs are designed to target a single step in a single disease and it is becoming increasingly clear that drugs that target multiple steps in multiple diseases work much better. Azurin and Laz appear to show the much desired promiscuity in targeting multiple, seemingly unrelated diseases such as cancer, malaria and AIDS / HIV, thus making them potentially valuable candidates as drugs.
Are proteins such as azurin and Laz the only anticancer agents from bacteria such as Pseudomonas and Neisseria? It has been known for a long time that DNA from Mycobacterium bovis BCG has antitumor property and a DNA fragment (or oligo deoxynucleotides) rich in the dinucleotide C and G (called CpG-rich DNA) can interfere in cancer growth through activation of the Toll-like receptor 9 (TLR9), thus activating production of cytokines and chemokines with anticancer activity. Azurin is secreted by P. aeruginosa when exposed to human breast cancer or melanoma cells as a weapon. Simultaneously, a CpG-rich piece of DNA is released from P. aeruginosa with anticancer activity, suggesting the use of multiple weapons by bacteria against cancer. The proteins such as azurin have structural similarity to human immunoglobulins, and demonstrate functional similarity to human antibodies as well. Most interestingly, azurin not only has cancerocidal activity, but it also interferes in oncogenic transformation, meaning that azurin potentially can prevent cancer recurrence. Azurin thus represents a candidate drug that not only has activity against cancer, HIV / AIDS,malarial parasite or the parasite that causes Toxoplasmosis but may interfere in the reappearance of cancer, making it a valuable potential drug in treating multiple diseases. While researchers normally study host-pathogen interactions, trying to develop inhibitors of such interactions to be used as anti-infective agents, very few people study pathogen – pathogen interactions where a bacterial pathogen such as P.aeruginosa elaborates azurin like proteins to prevent the entry and establishment of other pathogens including viruses, parasites or even cancer. We believe such promiscuous candidate drugs as azurin and Laz from pathogenic bacteria will be our next generation drugs to treat cancers, parasitic and viral infections.
Since my current interest is in the development of bacterial anticancer protein/peptide drugs, I have co-founded two companies to help bring such drugs to the bedside. One company in Chicago, CDG Therapeutics Inc. (http://www.cdgti.com), has sponsored clinical trials with a peptide derived from azurin termed p28. UIC holds many US and international patents on azurin/p28 that have been licensed exclusively to CDGTI. In two completed phase I trials, p28 demonstrated very little toxicity but significant beneficial effect in 15 stage IV cancer patients and 12 pediatric brain tumor patients (see the website http://www.cdgti.com) . A second company in India, Amrita Therapeutics (http://www.amritatherapeutics.com) is in the process of developing similar bacterial anticancer peptide drugs from a different bacterium and a different protein. Thus my current efforts are mostly in the area of translational research.