Rapamycin induces p-Akt in MM cells, while perifosine inhibits p-Akt
To confirm the effects of rapamycin signaling on MM cells, MM.1S cells were exposed to increasing concentrations of rapamycin for 2 hours. Rapamycin treatment resulted in a dose-dependent decrease of p-P70S6K. This was accompanied by an increase in phosphorylation of Akt at Ser473, starting at doses as low as 1 nM. Inhibition of p-P70S6K and activation of p-Akt were observed as early as 30 min after exposure of MM cells to rapamycin indicating that suppression of p-P70S6K and activation of Akt are early, concurrent, and lasting effects induced by rapamycin in MM.1S cells ().
Rapamycin induces p-Akt in MM cells, while perifosine inhibits Akt
We next examined the effects of perifosine on mTOR/Akt signaling in MM cells. MM.1S cells were cultured for 2 hours in the presence of increasing doses of perifosine (2.5–5 uM). Because perifosine was able to completely inhibit Akt phosphorylation at 5 uM, we next performed a time course (0.5 to 8 hours) to examine the effects of perifosine (5 uM) on Akt and P70S6K phosphorylation. Our data () demonstrates that perifosine inhibited Akt, without exhibiting evident effects on P70S6K phosphorylation in a dose- and time-dependent manner.
We next incubated MM.1S cells with rapamycin (10 nM), perifosine (5 uM), or the combination for the specified times to study effects on cell signaling and cytotoxicity. As shown in , rapamycin treatment resulted in increased p-Akt, which was overcome by the combination as early as 6 hours, associated with enhanced cytotoxicity at 48 hours ().
To determine whether rapamycin effects were cell line specific we tested other MM cell lines. Our data demonstrates activation of Akt in OPM1, OPM2, and U266 MM cells in the presence of rapamycin (10 nM) at 6 hours. Similar to MM1.S cells, the combination of rapamycin and perifosine abrogated Akt phosphorylation in OPM1, OPM2, and U266 cells and resulted in enhanced cytotoxicity with the combination treatment in all 3 MM cell lines (supplementary data
). Furthermore, 48 hour co-culture of MM.1S cells with rapamycin (10 nM) and the selective Akt kinase inhibitor Akti-½ (1.25 uM) potentiated rapamycin-induced cytotoxicity (supplementary data
), confirming the enhanced cytocidal effect with dual mTOR and p-Akt inhibition.
Using Chou-Talalay method, we examined possible additive or synergistic anti-proliferative effects of rapamycin and perifosine following 48 hours treatment in MM.1S cells. Doses below IC50 for rapamycin (1-10 nM) and perifosine (2.5-5 uM) were used for combination studies. Combination index (CI) ranged from 0.468 to 0.165, suggesting synergistic growth inhibitory activity ().
Rapamycin and perifosine overcome the growth and survival advantage conferred by IL-6, IGF-1 and BMSCs in MM.1S cells
Because of the critical role played by BMSCs and cytokines such as IL-6 and IGF-1 on the growth and survival of MM cells and their impact on the PI3K/Akt pathway in the context of drug resistance, we examined the effects of rapamycin and perifosine combination in the presence of cytokines and stroma. As shown in , IL-6 triggered Akt phosphorylation, which was inhibited when rapamycin and perifosine were combined. The suppression of p-Akt by rapamycin and perifosine after IGF-1 stimulation was not as robust, suggesting that once activated IGF-1 signaling strongly upregulates Akt activity and there might be other signaling circuits contributing to p-Akt phosphorylation. However, when combined, rapamycin and perifosine increased the cytotoxicity in IL-6- and IGF-1-stimulated MM.1S cells (). Similarly, the combination was studied in the context of BMSCs. Adherence of MM.1S cells to BMSCs triggered upregulation of p-Akt; the combination blocked this effect, resulting in p-Akt downregulation (). Furthermore, the proliferative advantage conferred by BMSCs was overcome by the combination, as demonstrated by [3H]-thymidine uptake and confirmed by CI=0.986.
Rapamycin and perifosine combination overcomes the growth and survival advantage in MM.1S cells conferred by IL-6 and IGF or BMSCs
Rapamycin-induced autophagy resulted in apoptosis when combined with perifosine
Since an increasing number of studies indicate that inhibition of mTOR results in induction of autophagy, we examined whether rapamycin treatment triggers autophagy in MM.1S cells. Because our data demonstrates rapamycin-induced downregulation of p-P70S6K as early as 30 min suggesting rapid mTOR inhibition, we first determined whether rapamycin treatment triggered early autophagy. Second, because of p-Akt’s ability to disinhibit mTOR (33
), we hypothesized that inhibition of rapamycin-induced p-Akt activity by the combination of rapamycin and perifosine might facilitate initiation of autophagy. MM.1S cells were exposed to rapamycin (10 nM), perifosine (5 uM), the combination, or media alone for 3 hours, and ultrastructural morphology of the cells were analyzed by electron microscopy. As seen in , rapamycin-treated cells exhibited morphological changes characteristic of autophagy with presence of single-and double-membrane limiting vesicles sequestering the cytosolic material, which were not evident in perifosine-treated cells. These were more abundant when rapamycin and perifosine were combined. These microscopic observations suggested that rapamycin results in autophagy in MM.1S cells at early time points, and that rapamycin-induced autophagy was enhanced when rapamycin and perifosine were combined.
Rapamycin-mediated autophagy resulted in apoptosis when combined with perifosine
To confirm rapamycin-induced autophagy and gain insights into the extent of increased autophagy triggered by the combination, we examined the effect of these drugs on localization of LC3, which serves as a marker of autophagy. We tested the effect of 3-hour treatment with rapamycin, perifosine, or both on localization of LC3 in MM.1S cells by immunofluorescence microscopy (). Untreated control cells exhibited diffuse distribution of LC3-associated green fluorescence, while rapamycin-treated MM.1S cells displayed a punctate pattern of LC3 immunostaining with increased fluorescence indicating co-localization with autophagosomes. Perifosine-treated cells expressed less intense and mostly perinuclear staining, while the combination demonstrated more focal LC3 green fluorescence predominantly in conglomerates, which suggests maturation of autophagic vacuoles.
Although autophagy is a response to various anticancer therapies, the extent to which autophagy contributes to cell death, known as type 2 or autophagic cell death, remains unclear. Shown in are morphological changes in MM.1S cells induced after 16 hours of treatment with rapamycin, perifosine, or the combination. Whereas untreated cells had normal nuclear and cytoplasmic morphology, rapamycin–treated cells developed typical features of autophagy with centrally condensed nuclear chromatin and numerous membranous vesicles. Higher magnification revealed double or multiple membrane boundaries surrounding cytoplasmic material and alternating with electron dense vesicles. Conversely, perifosine–treated cells manifested morphological characteristics of apoptosis, with nuclear condensation (pyknosis) and fragmentation (karyorhexis), cell shrinkage, plasma membrane blebbing, and vacuolization. Rapamycin and perifosine co-treatment resulted in morphological features of both autophagy and apoptosis, with evidence of double-membrane autophagolysosomes containing cytoplasmic fragments and disintegrated organelles typical of autophagy as well as condensation and margination of chromatin characteristic of apoptosis.
Given that rapamycin-perifosine co-treatment induced both apoptosis and autophagy features in MM.1S cells, we investigated the effect of this combination on apoptosis. As shown in , although rapamycin-induced caspase 8 cleavage, it did not result in significant apoptosis of MM cells at 24 or 48 hours. However perifosine resulted in apoptosis and necrosis of 30% of MM cells at 48 hours. The combination resulted in enhanced caspase-dependent apoptosis, manifested by increased caspase 3, 8, 9 and PARP cleavage ().
Since the combination of rapamycin and perifosine was able to activate both autophagy and apoptosis in MM cells, we next investigated whether these cell death-associated phenomena were interconnected and defined their role in rapamycin and perifosine combination-induced programmed MM cell death. We evaluated whether blocking of either apoptosis or autophagy would compromise rapamycin and perifosine combination induced cytotoxicity by assessing viability of MM.1S cells in the presence or absence of z-VAD-fmk or 3-MA pretreatment (supplementary data
). Neither blockade of autophagy nor inhibition of apoptosis rescued MM cells from death induced by the combination, suggesting that cell death resulted once either mechanism was initiated.
In silico rapamycin and perifosine combination study confirms the Akt/mTOR kinase downregulation, and activated caspases
For a more comprehensive understanding of the cellular mechanisms underlying the synergism of this combination we proceeded with in silico tumor cell modeling. The objective was to analyze the predictive effects of the mTOR inhibitor rapamycin and the AKT inhibitor perifosine on the key kinases up-regulated in cancer and on other major end points for cancer phenotypes of proliferation, survival, and tumor microenvironment. The in silico study was performed on the iC-PHYS™ Oncology platform. Various clinically important markers were observed and their levels quantitatively compared under conditions of untreated control, rapamycin alone (10 nM), perifosine alone (5 uM), or the combination.
The key marker values are presented as the percentage difference between control versus each drug alone or the combination (). The in silico study confirmed that rapamycin-induced mTOR/ATP inhibition associates with upregulated p-Akt. As expected, perifosine alone reduced Akt activity, but did not have any effect on mTOR kinase level. Meanwhile, the combination decreased both Akt and mTOR kinases (). Rapamycin alone had no effect on caspases activation, while perifosine, as expected, increased the activity of caspase 3, 6, 9, and the combination ultimately resulted in cumulative signaling effects ().
Rapamycin and perifosine combination effects on major cancer phenotypes by in silico analysis
Effects of nab-rapamycin and perifosine alone or in combination on MM tumor growth in vivo
We finally sought to establish whether our in vitro observations would translate to anti-MM activity in vivo using our MM murine xenograft model. Due to the poor water solubility of rapamycin, we studied nab-rapamycin as a promising candidate for our in vivo MM studies. We first evaluated the toxicity and anti-MM activity of nab-rapamycin treatment for 4-weeks in our MM xenografts SCID mouse model. Both intravenous daily (30 mg/kg/day) and 3x weekly (20 mg/kg/day and 40 mg/kg/day) administration of nab-rapamycin resulted in significant inhibition of MM tumor growth and increased the survival of animals (data not shown). To investigate whether combined treatment with nab-rapamycin and perifosine would augment the anti-MM activity of each agent alone, MM tumor bearing SCID mice were treated for 4 weeks with nab-rapamycin (10 mg/kg/day; n = 10 mice) by tail vein injections on days 1, 3 and 5 for 4 weeks, perifosine (125 mg/kg; n = 10 mice) via oral gavage on day 5 for 4 weeks, or combination (n = 10 mice), nab-rapamycin on days 1, 3, 5 and perifosine given on day 5, for 4 weeks). Combined treatment with nab-rapamycin and perifosine significantly inhibited the growth of MM cell xenografts compared to administration of solvent alone (). Although each drug as a single agent inhibited tumor expansion (P< 0.05), combined nab-rapamycin and perifosine induced tumor growth arrest, assessed by tumor growth inhibition index (TGI) of 90% at the end of treatment.
Combination of nab-rapamycin and perifosine significantly inhibits MM cell growth in vivo
Moreover, at 5 week follow-up after completion of nab-rapamycin or perifosine treatment, tumors started to regrow as early as 2 weeks. In contrast, all mice treated with the combination had smaller tumors, suggesting that therapeutic effects were maintained even after treatment was terminated.
Toxicity observed with the combination of nab-rapamycin and perifosine was evidenced by 20% weight loss at day 12 after initiation of treatment, which reversed after completion of treatment ().
The control and treated animals were maintained for their natural life span or sacrificed in the presence of a very large (≥ 1000 mm3 tumor volume) or ulcerated tumor. A significant survival advantage was observed when nab-rapamycin was combined with perifosine, as shown in . At day 61 after the beginning of treatment, only 10% of the animals survived in the control group versus 40% in each single-drug treated groups; in contrast, 80% of the animals were alive in the combination-treated mice. Moreover, 80% of mice in the combination–treated arm were still alive at day 75 following treatment initiation. There were no survivors in the control or monotherapy cohorts.
Given the therapeutic efficacy of nab-rapamycin and perifosine combination in our in vivo MM model, we next examined the associated histological events. Four mice were subjected to a similar in vivo study; mice were sacrificed and tumors collected after 1 week-treatment. As seen in , nab-rapamycin induced p-Akt in tumor tissue, which was inhibited when nab-rapamycin was combined to perifosine. LC3 immunohistochemical staining identified distinct patterns: LC-3 diffuse cytoplasmic expression (basal) in vehicle- and nab-rapamycin-treated tumors versus patchy-distribution staining in perifosine-treated tumor (). Interestingly, the combination-treated tumor showed increased LC3 staining in both diffuse and patchy patterns, along with more cleaved Caspase 3 and TUNEL-positive cells (). These findings therefore support our in vitro data showing amplification of both autophagy and apoptosis.
Ex vivo immunohistochemistry staining for p-Akt, LC3, TUNEL, and cleaved caspase 3 in MM xenografts