Colon cancer therapeutics
Nature weds chemistry 

Several years ago, Shrikant Anant, Ph.D., of Cancer Prevention, had just isolated the active ingredient from stone apple juice – an Ayurvedic remedy for irritable bowel syndrome – when, somewhat serendipitously, he observed firsthand its ability to kill assorted cancer cells cultured in the laboratory. Not knowing the mechanistic details involved, he decided that said ingredient’s modus operandi was worth a closer look. 

marmelin figure 
Chemical structure of marmelin.  

Stone apples hail from the Bael tree, an aromatic gum species native to India, and are among many natural compounds Anant has devoted one level of his research niche to investigating for anticancer activity. He renamed this intriguing ingredient “marmelin” after the tree species’ scientific handle, Aegle marmelos; it was otherwise known as HDNC, the shortened form of its unwieldy chemical identification. Then on faculty at the University of Oklahoma, Anant proceeded to figure out how marmelin extinguishes cancer cells, reporting in the October 2008 issue of Cancer Research that it triggers apoptosis – a form of programmed cell death necessary for regulating normal tissue development, and one that cancer cells often evade – by activating the inflammatory signaling molecule TNF-α and its receptor protein, TNFR1.

Separate probing of his research niche’s second level – cancer stem cells, a small group thought to play critical roles in maintaining or forming new tumors – had led Anant to jointly discover with his Oklahoma colleague Courtney Houchen, M.D., that DCLK-1, a member of the kinase family and first identified in the brain, is also a “fingerprint” that marks cancer stem cells in the colon. Knocking down the gene for DCLK-1 in colon cancer cells completely suppressed tumor development when those cells were grafted in mice. The game was on to find some non-laboratory means of suppressing DCLK-1, preferably via a specific molecule or compound.

Anant and his research group, notably protégés Dharmalingam Subramaniam, Ph.D., and Deep Kwatra, Ph.D., began closely examining DCLK-1’s enzyme structure, and it was then that two previously independent findings dovetailed: marmelin appeared to be a good fit for blocking DCLK-1’s catalytic “pocket,” or substrate binding site. But the compound itself failed to halt DCLK-1’s activity, so the researchers turned to Subhash Padhye, Ph.D., a medicinal chemist at the University of Pune in India, for help.

“Some natural compounds have what are called “privileged structures,” or the potential to act as scaffolds that can be modified to possess specific drug properties,” Anant says. “We figured marmelin would be a good candidate here.” Others in this category include curcumin, the active ingredient of turmeric; and thymoquinone, from black cumin seeds. Any one compound’s medicinal uses represent ongoing evolution over thousands of years: what Padhye describes as its “genealogical pathway.” Staying one jump ahead of evolution, he and others of his ilk have sped up the process by first predicting these genealogical moves in compounds of interest, then altering their structures accordingly. After subjecting marmelin – itself the product of seven alterations by Mother Nature, spanning some 210,000 years – to this guessing-game treatment, several new possibilities emerged for Anant and his group to pit against DCLK-1. 

The researchers proceeded to combine DCLK-1 and its substrate, glycogen synthase peptide, with these iterations of marmelin and the energy needed (ATP molecules) for a catalytic reaction. Avoiding radioactively-labeled ATP as a readout, they instead added luciferin – a light-emitting compound in bioluminescent organisms – and its enzyme, luciferase, to the mix toward the end. Normally, DCLK-1 acting on its substrate chews up energy, leaving little for luciferase’s use and a corresponding drop in light. Under these conditions, muzzling DCLK-1 with a competitive inhibitor should produce a net effect of increased light; Anant found that this was indeed the case with a particular adaptation of marmelin, abbreviated as THB, from Padhye’s laboratory. 

Anant and his group then put THB through its paces in assorted proof-of-principle studies, noting that it halts the proliferation of colon cancer cells cultured in a microenvironment closely reminiscent of their normal milieu, and reduces total tumor mass in mice. Importantly, it also blocks the growth of colon cancer stem cells, which are readily visible to the naked eye as round pinhead-sized clumps, or spheroids, in the laboratory. The researchers showed that THB gags unruly signaling manned by the protein Notch, which dysfunctional molecular babble otherwise drives tumor resilience and progression. They also found that THB stifles another protein, PXR, which is often inadvertently switched on by chemotherapy agents and helps promotes drug resistance. 

Buoyed by these encouraging observations, Anant has just secured RO1 funding from the National Cancer Institute to carry out preclinical animal studies on both this marmelin derivative and THB conjugated to cyclodextrin, a sugar compound that renders it water-soluble. Scott Weir, Ph.D., of Drug Discovery, Development and Therapeutics, is on board as co-principal investigator to oversee the pharmacological aspects of these studies – determining the appropriate dose, route of administration and potential side effects, among other details. Within the next five years or so, barring unforeseen complications, Anant and his crew hope to amass enough solid data to warrant an early phase clinical trial in colon cancer: essentially, nudging marmelin that much further along its bench-to-bedside journey. 

Funding sources for this research

  • NIH RO1 CA109269: “Dietary Prevention of Cancer”

  • NIH RO1 CA182872: “Novel Dual Notch/PXR Targeting for Colon Cancer Therapy”

Related publications

  • Subramaniam D, Ramalingam S, Houchen CW, Anant S, “Cancer stem cells: a novel paradigm for cancer prevention and treatment.” Mini Rev Med Chem (May 2010). 
  • May R, Sureban SM, Hoang N, Riehl TE, Lightfoot SA, Ramanujam R, Wyche JH, Anant S, Houchen CW, “Doublecortin and CaM kinase-like-1 and leucine-rich-repeat-containing G-protein-coupled receptor mark quiescent and cycling intestinal stem cells, respectively.” Stem Cells (Oct 2009). 
  • Subramaniam D, Giridharan P, Murmu N, Shankarnarayanan NP, May R, Houchen CW, Ramanujam RP, Balakrishnan A, Vishwakarma RA and Anant S, “Activation of apoptosis by 1-hydroxy-5, 7-dimethoxy-2-napthaelene-carboxaldehyde, a novel compound from Aegle marmelos.” Cancer Research (Oct 2008).