Serum thymidine kinase 1 activity as a pharmacodynamic marker of cyclin-dependent kinase 4/6 inhibition in patients with early-stage breast cancer receiving neoadjuvant palbociclib.

BACKGROUND: Thymidine kinase 1 (TK1) is a cell cycle-regulated enzyme with peak expression in the S phase during DNA synthesis, and it is an attractive biomarker of cell proliferation. Serum TK1 activity has demonstrated prognostic value in patients with early-stage breast cancer. Because cyclin-dependent kinase 4/6 (CDK4/6) inhibitors prevent G1/S transition, we hypothesized that serum TK1 could be a biomarker for CDK4/6 inhibitors. We examined the drug-induced change in serum TK1 as well as its correlation with change in tumor Ki-67 levels in patients enrolled in the NeoPalAna trial (ClinicalTrials.gov identifier NCT01723774). METHODS: Patients with clinical stage II/III estrogen receptor-positive (ER+)/HER2-negative breast cancer enrolled in the NeoPalAna trial received an initial 4 weeks of anastrozole, followed by palbociclib on cycle 1, day 1 (C1D1) for four 28-day cycles, unless C1D15 tumor Ki-67 was > 10%, in which case patients went off study owing to inadequate response. Surgery occurred following 3-5 weeks of washout from the last dose of palbociclib, except in eight patients who received palbociclib (cycle 5) continuously until surgery. Serum TK1 activity was determined at baseline, C1D1, C1D15, and time of surgery, and we found that it was correlated with tumor Ki-67 and TK1 messenger RNA (mRNA) levels. RESULTS: Despite a significant drop in tumor Ki-67 with anastrozole monotherapy, there was no statistically significant change in TK1 activity. However, a striking reduction in TK1 activity was observed 2 weeks after initiation of palbociclib (C1D15), which then rose significantly with palbociclib washout. At C1D15, TK1 activity was below the detection limit (<20 DiviTum units per liter Du/L) in 92% of patients, indicating a profound effect of palbociclib. There was high concordance, at 89.8% (95% CI: 79.2% - 96.2%), between changes in serum TK1 and tumor Ki-67 in the same direction from C1D1 to C1D15 and from C1D15 to surgery time points. The sensitivity and specificity for the tumor Ki-67-based response by palbociclib-induced decrease in serum TK1 were 94.1% (95% CI 86.2% - 100%) and 84% (95% CI 69.6% -98.4%), respectively. The κ-statistic was 0.76 (p < 0.001) between TK1 and Ki-67, indicating substantial agreement. CONCLUSIONS: Serum TK1 activity is a promising pharmacodynamic marker of palbociclib in ER+ breast cancer, and its value in predicting response to CDK4/6 inhibitors warrants further investigation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01723774. Registered on 6 November 2012.

Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.

Bioconjugation by native chemical tagging of C-H bonds.

A general C-H functionalization method for the tagging of natural products and pharmaceuticals is described. An azide-containing sulfinate reagent allows the appendage of azidoalkyl chains onto heteroaromatics, the product of which can then be attached to a monoclonal antibody by a "click" reaction. This strategy expands the breadth of bioactive small molecules that can be linked to macromolecules in a manner that is beyond the scope of existing methods in bioconjugation to permit tagging of the "seemingly untaggable".

Antiproliferative acetogenins from a Uvaria sp. from the Madagascar dry forest.

Investigation of the endemic Madagascan plant Uvaria sp. for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of two new acetogenins. The structures of these two compounds were elucidated on the basis of analysis of their 1D and 2D NMR spectra, circular dichroism, and mass spectrometric data, together with chemical modification. The two acetogenins display weak antiproliferative activity against the A2780 ovarian cancer, the A2058 melanoma, and the H522 lung cancer cell lines.

Cardenolides of Leptadenia madagascariensis from the Madagascar dry forest.

Investigation of the endemic Madagascar plant Leptadenia madagascariensis Decne. (Apocynaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the four new cardenolides 1-4. The structure elucidations of these compounds were based on analyzes of their 1D and 2D NMR spectra and mass spectrometric data. The cardenolides were strongly antiproliferative to the A2780 ovarian cancer cell line, with IC(50) values of 0.18, 0.21, 0.17, and 0.29µM line, and to the H460 human lung cancer cell line, with IC(50) values of 0.16, 0.68, 0.37, and 0.48µM, respectively.

Isolation and synthesis of antiproliferative eupolauridine alkaloids of Ambavia gerrardii from the Madagascar Dry Forest.

Investigation of the Madagascan endemic plant Ambavia gerrardii for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the three new alkaloids 8-hydroxyeupolauridine (1), 9-methoxyeupolauridine 1-oxide (2), and 11-methoxysampangine (3) and the three known alkaloids 4-6. The structures of 1 and 2 were confirmed by synthesis. Compounds 3, 4, and 6 showed moderate to good antiproliferative activities, with IC50 values of 10.3, 3.5, and 0.60 µM, respectively, against the A2780 human ovarian cancer cell line and with IC50 values of 0.57, 1.77, and 0.58 µM, respectively, against the H460 human lung cancer cell line.

Antiproliferative compounds from Pongamiopsis pervilleana from the Madagascar Dry Forest.

Bioassay-guided fractionation of an ethanol extract of the roots of the endemic Malagasy plant Pongamiopsis pervilleana led to the isolation of the three new compounds (2'R)-4'-hydroxyemoroidocarpan (1), pongavilleanine (3), and epipervilline (4) together with two known compounds, identified as emoroidocarpan (2) and rotenolone (5). The structures of all compounds were determined by physical, chemical, and spectroscopic evidence. The stereochemistry at C-2' of the previously reported compound emoroidocarpan was determined to be R by the observation of a negative Cotton effect at 474 nm in the CD spectrum of its osmate ester derivative. Compounds 2-5 displayed moderate antiproliferative activity against the A2780 human ovarian cancer cell line, and rotenolone also showed micromolar antiproliferative activity toward the breast cancer BT-549, prostate cancer DU 145, NSCLC NCI-H460, and colon cancer HCC-2998 cell lines.

Antiproliferative cardenolide glycosides of Elaeodendron alluaudianum from the Madagascar Rainforest.

Bioassay-guided fractionation of an ethanol extract of a Madagascar collection of Elaeodendron alluaudianum led to the isolation of two new cardenolide glycosides (1 and 2). The (1)H and (13)C NMR spectra of both compounds were fully assigned using a combination of 2D NMR experiments, including (1)H-(1)H COSY, HSQC, HMBC, and ROESY sequences. Both compounds 1 and 2 were tested against the A2780 human ovarian cancer cell line and the U937 human histiocytic lymphoma cell line assays, and showed significant antiproliferative activity with IC(50) values of 0.12 and 0.07 microM against the A2780 human ovarian cancer cell line, and 0.15 and 0.08 microM against the U937 human histiocytic lymphoma cell line, respectively.

Antiproliferative triterpenoid saponins of Dodonaea viscosa from the Madagascar dry forest.

Bioassay-guided fractionation of an EtOH extract obtained from the roots of the Madagascan plant Dodonaea viscosa led to the isolation of two new antiproliferative oleanane-type triterpenoid saponins, dodoneasides A and B (1 and 2). The structures of these two new compounds were elucidated using 1D and 2D NMR experiments and mass spectrometry. Compounds 1 and 2 showed antiproliferative activity against the A2780 human ovarian cancer cell line with IC(50) values of 0.79 and 0.70 muM, respectively.

Euphane triterpenoids of Cassipourea lanceolata from the Madagascar rainforest.

Fractionation of an ethanol extract of a Madagascar collection of the leaves and fruit of Cassipourea lanceolata Tul. led to the isolation of three euphane triterpenoids 1-3. The (1)H and (13)C NMR spectra of all compounds were fully assigned using a combination of 2D NMR experiments, including COSY, TOCSY, HSQC (HMQC), HMBC and ROESY sequences. The three compounds showed weak antiproliferative activities against the A2780 human ovarian cancer cell line, with IC(50) values of 25, 25 and 32 microM, respectively.

Tubulin-based antimitotic mechanism of E7974, a novel analogue of the marine sponge natural product hemiasterlin.

E7974 is a synthetic analogue of the marine sponge natural product hemiasterlin. Here, we show that E7974, such as parental hemiasterlin, acts via a tubulin-based antimitotic mechanism. E7974 inhibits polymerization of purified tubulin in vitro with IC(50) values similar to those of vinblastine. In cultured human cancer cells, E7974 induces G(2)-M arrest and marked disruption of mitotic spindle formation characteristic of tubulin-targeted anticancer drugs. Extensive hypodiploid cell populations are seen in E7974-treated cells, indicating initiation of apoptosis after prolonged G(2)-M blockage. Consistent with this observation, E7974 induces caspase-3 activation and poly ADP ribose polymerase cleavage, typical biochemical markers of apoptosis. Only a short cellular exposure to E7974 is sufficient to induce maximum mitotic arrest, suggesting that E7974's antitumor effects in vivo may persist even after blood levels of the drug decrease after drug administration. Interactions of E7974 with purified tubulin were investigated using two synthetic tritiated photoaffinity analogues incorporating a benzophenone photoaffinity moiety at two different positions of the E7974 scaffold. Both analogues preferentially photolabeled alpha-tubulin, although minor binding to beta-tubulin was also detected. E7974 thus seems to share a unique, predominantly alpha-tubulin-targeted mechanism with other hemiasterlin-based compounds, suggesting that, unlike many tubulin-targeted natural products and related drugs, the hemiasterlins evolved to mainly target alpha-tubulin, not beta-tubulin subunits.

Antiproliferative limonoids of a Malleastrum sp. from the Madagascar rainforest.

Bioassay-guided fractionation of an ethanol extract of a Malleastrum sp. afforded three new limonoids, malleastrones A-C ( 1- 3), respectively. Each limonoid contained a rare tetranortriterpenoid skeleton. Structure elucidation of the isolates was carried out by analysis of one- and two-dimensional NMR and X-ray diffraction data. The novel isolates 1 and 2 were tested for antiproliferative activity against a panel of cancer cell lines and exhibited IC 50 values ranging from 0.19 to 0.63 microM.

Antiproliferative compounds of Artabotrys madagascariensis from the Madagascar rainforest.

Bioassay-guided fractionation of an ethanol extract of Artabotrys madagascariensis led to the isolation of the new compound artabotrene (1), two butenolides (2 and 3), and the tetracyclic triterpene polycarpol (4). Structure elucidation was determined on the basis of one and two-dimensional NMR, and absolute configuration of compounds 2-4 was verified by analysis of CD and optical rotation spectra. Two of the isolates, melodorinol (2) and acetylmelodorinol (3), were found to display antiproliferative activity against five different tumour cell lines with IC50 values ranging from 2.4 to 12 microM.

Antiproliferative cardenolides of an Elaeodendron sp. from the Madagascar rain forest(1).

Bioassay-guided fractionation of an ethanol extract obtained from the Madagascar plant Elaeodendron sp. led to the isolation of two new cardenolides, elaeodendrosides T and U (1 and 2). The structures of the new compounds were elucidated using 1D and 2D NMR experiments and mass spectrometry. Compounds 1, 3, 4, and 5 showed significant antiproliferative activity against A2780 human ovarian cancer cells with IC50 values of 0.085, 0.019, 0.19, and 0.10 microM, respectively, while compounds 2 and 6 were less active.