Among cancer treatments, targeted chemotherapies are valued for minimizing side effects and whole-body toxicity, while delivering damage to malignant cells. To achieve these goals, a chemotherapeutic agent must selectively bind to or target tumor cells or, alternatively, become active in tumor cells but remain inactive elsewhere in a patient’s body.
Researchers at St. Mary’s College of Maryland recently focused their attention on the chemotherapeutic use of an azo- compound (azo- compounds contain a nitrogen-nitrogen double bond) that they designed to convert from an inactive to an active form when exposed to blue or near-UV wavelength light. The compound studied, azo-combretastatin A4, was synthesized by Craig N. Streu, PhD, assistant professor of chemistry and biochemistry at St. Mary’s College.
“I’d been reading some papers and saw that people were using these azo- compounds for all sorts of things,” remembers Streu. “They were turning genes on, turning genes off. And I thought: ‘This structure looks like a lot of small-molecule drugs: What types of small-molecule drugs can I incorporate these things into?’”
After working with azo-resveratrol (resveratrol is the “longevity compound” in red wine), Streu turned his attention to the compound combretastatin A4, whose structure resembles resveratrol and
Light activatable azo-combretastatin A4 (Azo-CA4) was synthesized based on the structures of commercial tubulin inhibitors colchicine and combretastatin A4 (CA4). Azo-CA4, activated with a $7 flashlight, was shown to be an effective tubulin inhibitor compared to the dark Azo-CA4 control.
– even more interestingly – is similar to colchicine. Colchicine, first isolated from the autumn crocus in 1820, has long been recognized for its medicinal uses. Among its effects, colchicine is known to impair cell division by disrupting a cell’s microtubules.
But, back to combretastatin A4 … Drugs bind to their targets much like keys fit into locks, so changing the shape of a drug often alters the effect on its biological target. “The active form of azo-combretastatin A4 has the double bond in the cis conformation,” explained Streu. “That’s the photioisomerizable bond, and the shape of the molecule changes around that nitrogen-nitrogen double bond: In one conformation [trans] the molecule is really flat, in the other conformation [cis], the molecule is twisted.”
When azo-combretastatin A4 is converted from its trans (inactive) to cis (active) form by exposure to blue or UV light, the active form has the same effect as colchicine: It impairs normal cell division by binding to tubulin molecules, preventing them from assembling into microtubules.
“Tubulin polymerizes and depolymerizes, and that polymerization makes long chains that are really important for mitosis, the cell division process,” said Streu. “If you can interfere with the way these long chains of tubulin are assembled, you interfere with the cell-division process.”
But would this work in living cells? To investigate the effect of azo-combretastatin A4 on living cells, Pamela Mertz, PhD, associate professor of chemistry and biochemistry at St. Mary’s College, tested the compound in HeLa cell cultures. Widely used in research, HeLa cells were originally derived from an aggressively growing human cervical cancer.
“A lot of people work with HeLa cells because they’re very easy in terms of working with a cell culture. They’re very hardy cells; they’re not very delicate … HeLa has the advantage of being a stable cell line and not very finicky. They grow and divide and are quite happy,” explained Mertz.
The goal in this study, however, was to determine how activated azo-combretastatin A4 would affect these “happy” HeLa cells. “Tubulin is an important protein that forms the cytoskeleton of the cell, which is very important in terms of the overall architecture of holding the cell together,” Mertz noted.
“A number of different types of chemotherapeutic drugs have been developed specifically to target tubulin. While all cells need it, the idea there, of course, is that you’re trying to target rapidly dividing cells in terms of cancer and hopefully knock those out. … One advantage of trying to develop these more targeted compounds, that you can activate with light, is that you’d have a tool for being more specific, targeting a particular area in the body vs. the whole body.”
The researchers cultured HeLa cells for five days in the presence of various concentrations of azo-combretastatin A4. One group of cultures was grown in the absence of light, another group was exposed to 10 seconds of 400 nm light (violet: 400-450 nm; UV: 300-400 nm) every 30 minutes. Light-exposed HeLa cell cultures showed complete cell death at azo-combretastatin A4 concentrations as low as 500 nM. Dark-grown cultures displayed little or no toxic effects, even with azo-combretastatin A4 concentrations as high as 100 uM.
Thus, when tested in cell cultures, photoisomerized azo-combretastatin A4 was at least 200 times more toxic to malignant cells than was its inactive form. Although this was not a clinical study, one obvious question is whether photoisomerization of azo-combretastatin A4 would be feasible for tumors deep in the body. How could light be administered to activate the molecule?
“There are whole groups who work on this problem,” said Streu. “They’ve made little implantable electrode LEDs for photodynamic therapy … We already implant all sorts of things for timed delivery of chemotherapeutics, and one of the things they implant are little LED lights. We expect that this will be more common as more phototherapeutics are developed.”
Both Dr. Streu and Dr. Mertz also praised the involvement of undergraduate researchers who contributed to this study. “In terms of this paper … almost all of the data were generated by undergraduates,” said Mertz. “If you look at the first four authors, they’re all undergraduates at St. Mary’s … They really are the ones performing the experiments and they really have ownership over it by the time they finish a project like this.”