Some of the most clinically effective antitumor drugs developed over the past 25 years are those that target cellular microtubules [1
]. Microtubule-targeting drugs are often classified as microtubule stabilizers, which include the taxanes and epothilones, or destabilizers, represented by the vinca alkaloids and combretastatin A4. These designations, stabilizer or destabilizer, refer to the ability of these drugs to cause striking changes in the microtubule structures of interphase cells. The extensive work of Jordan and Wilson showed that at lower, clinically relevant concentrations, microtubule stabilizers and microtubule destabilizers have the same general mechanism of action: they inhibit microtubule dynamics leading to apoptosis [2
Paclitaxel and docetaxel are taxanes that effectively treat breast, ovarian, prostate and non-small-cell lung cancer [1
]. Although the taxanes are arguably some of the most valuable chemotherapeutic agents available today, intrinsic and acquired drug resistance limit their anticancer actions. The identification of new microtubule stabilizers that can overcome taxane resistance mechanisms could provide significant breakthroughs in the treatment of cancer [6
]. The search for new classes of microtubule-targeting agents has been successful, and the epothilones and discodermolide have been evaluated in the clinic. In October 2007, ixabepilone (Ixempra™) was the first epothilone approved for the treatment of multidrug-resistant breast cancer [7
]. Discodermolide was evaluated in a Phase I trial but failed to advance due to lung toxicity [8
]. Other chemically diverse classes of microtubule stabilizers, including the laulimalides, peloruside A and the taccalonolides, have potential to be useful in cancer therapy.
We previously discovered the taccalonolides, a unique class of microtubule stabilizers that are highly acetylated steroids isolated from the tropical plant Tacca chantrieri
]. In cells, the effects of the taccalonolides (taccas) are almost identical to the effects of the taxanes. The most abundant taccalonolides isolated, A and E (taccas A and E), cause an increase in the density of interphase microtubules and shift cellular tubulin to the polymerized form. Consistent with the effects of other microtubule stabilizers, the taccas induce the formation of abnormal mitotic spindles leading to mitotic arrest, Bcl-2 phosphorylation and initiation of apoptosis [9
]. However, certain findings indicate that the taccas have a different mechanism of action as compared with other microtubule stabilizers [10
]. Unlike other compounds that are able to exert taxane-like effects in cells, the taccas do not robustly stimulate the polymerization of purified bovine brain tubulin or microtubule protein [10
]. Further studies indicate that the taccas do not bind to the taxane-binding site of tubulin [10
]. Studies are ongoing to identify the cellular binding site of the taccas and the mechanism of their antimitotic activity.
A significant proportion of cancer cell lines and tumors are multidrug-resistant due to expression of ABC (ATP-binding cassette) transporters. These transporters have multiple physiological roles, including the capacity to extrude xenobiotics from cells in an ATP-dependent manner. The MDR1
gene product P-glycoprotein (Pgp, ABCB1) functions in the cellular export of a wide spectrum of compounds, including complex natural products including the taxanes [11
]. Expression of Pgp in tissues such as the intestinal tract, liver and kidney contributes to the intrinsic taxane resistance of epithelial tumors derived from these tissues. In cells selected in vitro
and in vivo
for resistance to agents including the taxanes, upregulation of Pgp expression often leads to diminished intracellular drug accumulation and attenuated cytotoxic effects [11
]. Tumors in genetic murine models of breast cancer (Brca 1−/−, p53−/−
) that are initially sensitive to doxorubicin and docetaxel develop resistance following exposure to these drugs, which is attributable to expression of Pgp [13
]. Clinically, expression of Pgp in both hematological and solid tumors has been reported to be associated with poor treatment response and subsequent treatment failure [12
]. The overwhelming lack of success of Pgp inhibitors in the clinic indicates that the identification of agents that are not susceptible to Pgp-mediated resistance will be a crucial feature of new microtubule stabilizers [6
]. Hence, the anticancer efficacy of ixabepilone in multidrug-resistant breast cancer might relate to its ability to circumvent export by Pgp. Our previous work using the multidrug-resistant NCI/ADR cell line suggested that the taccas A and E are not transported by Pgp [9
]. In this study, we further explore the efficacy of the taccas in cells transduced with a Pgp-expression vector compared with an isogenic parental cell line.
In addition to Pgp, other resistance factors may affect the efficacy of microtubule-targeting agents. Expression of the ABC transporter MRP7 (multidrug-resistance protein 7; ABCC10) has recently been identified as one such factor. MRP7 is an ABC transporter with little structural homology to other MRP family members, although it shares their capacity to transport amphipathic anions [18
]. Noted substrates for MRP7 include 17β-estradiol-(17-β-D-glucuronide), docetaxel, paclitaxel, vinblastine and vincristine [19
]. MRP7 is normally expressed in stomach, colon, kidney, brain, pancreas, liver, lung, ovary and lymph nodes and has been detected in cancer cell lines and tumor samples [19
]. In cancer cell lines, the overexpression of MRP7 leads to 10-fold resistance to docetaxel and lower but significant resistance to paclitaxel and the vinca alkaloids [20
]. Additionally, MRP7 expression was found to be induced in non-small-cell lung cancer cell lines upon paclitaxel treatment. MRP7 expression levels correlated with both paclitaxel accumulation and sensitivity [23
]. These findings suggest that the ability of novel chemotherapeutic agents to circumvent MRP7-mediated efflux may provide a significant advantage for the treatment of cancer.
Overexpression of the βIII isotype of tubulin [reviewed in 24
] has been established as another clinically relevant resistance factor for the taxanes. Microtubules are formed of polymers of α/β heterodimers. Seven α-tubulin isotypes and nine β-tubulin isotypes have been described and tubulin isotype distribution is highly tissue specific. Under normal circumstances βIII-tubulin is found predominantly in neuronal tissues. However, in cancer cell lines and human tumors, the expression of βIII-tubulin has been implicated in drug resistance to microtubule-targeting drugs including the taxanes [24
]. Additionally, expression of βIII is often observed in cell lines made resistant to the taxanes by serial drug exposure [24
]. Incorporation of βIII into microtubules decreases microtubule dynamics [25
] and the removal of βIII from bovine brain tubulin results in microtubules that are more dynamic and more sensitive to paclitaxel [26
]. Inducible expression of βIII-tubulin has also been shown to diminish the ability of paclitaxel to inhibit microtubule dynamics in cells [27
]. Depletion of βIII by siRNA-mediated knockdown increases the sensitivity of cell lines to microtubule binding agents, further demonstrating the link between βIII-tubulin expression and drug resistance [28
Clinically, a number of studies indicate that the expression of the βIII isotype is associated with a lack of response to taxanes and poor clinical prognosis [24
]. In four clinical trials of non-small-cell lung cancer, the expression of βIII-tubulin was associated with resistance to a therapeutic regimen that included a taxane or vinorelbine, but not gemcitabine/cisplatin-based therapy [24
]. The correlation between high βIII-tubulin levels and resistance to taxane-based therapies has also been shown in ovarian, breast and gastric cancers, suggesting that the expression of this tubulin isotype might be a predictive marker for the success or failure of tubulin-targeting therapies.
Multidrug resistance is clearly multifactorial and often limits the success of chemotherapy. It is clear that alterations in the β-tubulin isotype composition and expression of specific ABC transporters, including MRP7 and Pgp, can lead to resistance to the microtubule-stabilizing drugs currently in clinical use. The successes and failures of the taxanes have prompted intense drug discovery efforts to find new classes of microtubule stabilizers. Here, we characterize the activity of the taccas in a variety of taxane-resistant cell lines and one murine tumor. We demonstrate that that taccas are not susceptible to multidrug resistance associated with overexpression of Pgp, MRP7 or the βIII-tubulin isotype. These findings indicate that taccas may have advantages over other microtubule-targeting agents and support their future preclinical evaluation.