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Keywords:

  • multidrug resistance;
  • P-glycoprotein;
  • hyaluronan oligosaccharides;
  • hematological malignancies;
  • PI3K/Akt

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Multidrug resistance (MDR) is one of the main reasons for failure of cancer therapy. It may be mediated by overexpression of ATP-dependent efflux pumps or by alterations in survival or apoptotic pathways. Fragments generated by enzymatic degradation of hyaluronan (oHA) were able to modulate growth and cell survival and sensitize MDR breast cancer cells to cytotoxic drugs. In this work the relationship between oHA and MDR in lymphoid malignancies was analyzed using murine lymphoma cell lines resistant to doxorubicin (LBR-D160) or vincristine (LBR-V160) and a sensitive line (LBR-). After oHA treatment, higher apoptosis levels were observed in the resistant cell lines than in the sensitive one. Besides, oHA sensitized LBR-D160 and LBR-V160 to vincristine showing increased apoptosis induction when used in combination with vincristine. Native hyaluronan failed to increase apoptosis levels. As different survival factors could be modulated by hyaluronan, we investigated the PI3K/Akt pathway through PIP3 production and phosphorylated Akt (p-Akt) and survivin expression was also evaluated. Our results showed that oHA decreased p-Akt in the 3 cell lines while anti-CD44 treatment abolished this effect. Besides, survivin was downregulated only in LBR-V160 by oHA. When Pgp function was evaluated, we observed that oHA were able to inhibit Pgp efflux in murine and human resistant cell lines in a CD44-dependent way. In summary, we report for the first time that oHA per se modulate MDR in lymphoma cells by decreasing p-Akt as well as Pgp activity, thus suggesting that oHA could be useful in combination with classical chemotherapy in MDR hematological malignancies. © 2007 Wiley-Liss, Inc.

The molecular mechanisms by which anticancer drugs fail to kill cancer cells have been widely studied; however, they are not completely understood yet. Multidrug resistance (MDR), one of the major obstacles to effective chemotherapy, may be mediated by overexpression of ATP-dependent efflux pumps such as phospho-glycoprotein (Pgp), reduced drug uptake, activation of detoxifying systems, or alterations in survival or apoptotic pathways.1 Human Pgp (MDR1) and its rodent homologues mdr-1 and mdr-3 are conserved proteins that translocate antitumor agents across the plasma membrane of resistant cancer cells, reducing intracellular drug concentrations to sublethal levels.2 Pgp-mediated drug efflux can be modulated by MDR inhibitors, which block transport in a competitive or noncompetitive way. Although many Pgp inhibitors (including verapamil, cyclosporin A and PSC833) are able to sensitize drug-resistant cell lines to antitumor agents in vitro, they have not always been useful in clinical trails since they appear to be either toxic or ineffective in vivo. Nowadays research is focused on the “fourth generation” inhibitors among old drugs, such as disulfiram or herbal components, which have been shown to modulate Pgp in vitro and could be used in safety doses in vivo.3

The balance between apoptotic and survival signals determines the susceptibility of cells to apoptosis. Since many chemotherapeutic drugs exert their cytotoxic effects by inducing apoptosis, alterations in cell survival and apoptotic signaling pathways appear to be important in MDR. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a central role in oncogenesis by regulating several biological processes such as cell survival and proliferation, apoptosis, cell growth, angiogenesis, tumor invasion and metastasis. PI3K activates Akt by phosphorylation. Once activated, phosphorylated Akt (p-Akt) phosphorylates multiple proteins implicated in cellular processes leading to induction of cell survival and inhibition of apoptosis. Akt overexpression is a frequent molecular alteration in human malignancies closely associated with chemoresistance, and it therefore constitutes a critical target for cancer intervention.4, 5

Survivin, one of the members of the inhibitor of apoptosis protein (IAP) family is a protein that suppresses apoptosis by inhibition of caspases. This protein is normally expressed in embryonic and fetal organs but not in adult differentiated tissues. However, several reports have demonstrated its expression in human tumors and enhanced survivin levels have been correlated with tumor progression.6 Besides, it has been recently shown that Akt may regulate survivin expression in neuroblastoma.7

Hyaluronan (HA) is a large, linear glycosaminoglycan with a molecular weight ranging from 105 to 107 Da. It is the major component of the mammalian extracellular matrix (ECM) where it has a structural function influencing hydration and physical properties of tissues.8 Besides, upon interaction with CD44 or RHAMM cell surface receptors, HA is able to modulate cell-signaling pathways.9 Increased levels of HA have been found in the ECM within tumors and have been related with tumor progression and metastasis.10, 11, 12, 13 However, HA oligosaccharides (oHA)—fragments generated by enzymatic degradation of hyaluronan—can stimulate dissimilar tumor behavior.14 It has been described that perturbation of hyaluronan-cell interactions by treatment with oHA suppresses PI3K/Akt cell survival signaling pathway in breast carcinoma cell lines, induces apoptosis and reduces tumor growth in vivo.15, 16 Moreover, oHA sensitize multidrug resistant breast tumor cells to a variety of chemotherapeutic agents while HA enhances MDR.17 Recent investigations have shown that oHA reduce constitutive MDR-1 expression in MDR breast tumor cells; in contrast, HA induces MDR-1 expression in drug sensitive cells.18 Although many studies have been performed in carcinoma models, little is known about the role of oHA and native HA on MDR in hematological malignancies.

The aim of this work was to analyze the relationship between oHA and MDR in murine lymphoma cell lines resistant to doxorubicin (LBR-D160) or to vincristine (LBR-V160) obtained previously in our laboratory.19 The ability of oHA to induce apoptosis and to sensitize MDR cells to antineoplasic drugs as well as the possible mechanisms involved were evaluated. Besides, modulation of Pgp activity and mdr-3 expression by oHA were also analyzed. Our results showed that oHA were able to induce higher apoptosis levels in murine lymphoma resistant cell lines (LBR-D160 and LBR-V160) than in the sensitive line (LBR-). Treatment with oHA decreased Akt phosphorylation as well as survivin levels. We also found that oHA sensitized both LBR-D160 and LBR-V160 to vincristine (VCR). In addition, we described for the first time that oHA were able to block Pgp function in murine lymphoma resistant as well as in human leukemic cell lines.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Reagents

Recombinant high (HMW 1.5–1.8 × 106 Da) and low (LMW 1–3 × 105 Da) molecular weight HA (CPN spol.s.r.o Czech Republic) were kindly supplied by Farmatrade (Argentina). Anti-CD44 (KM81) was kindly provided by Dr. K. Mikecz. Hyaluronidase and LY294002 were purchased from Calbiochem (La Jolla, CA). VCR was kindly provided by Filaxis Pharmaceuticals S.A. Argentina and DOX by Gador Pharmaceuticals Argentina. Antibodies against p-Akt, Akt, survivin, actin, rat IgG2a (isotype control), anti-rabbit secondary horseradish peroxidase, anti-goat secondary horseradish peroxidase, and Western Blotting Luminol reagent were purchased from Santa Cruz Biotechnology (USA). Antibody against PIP3 was from Echelon Biosciences and Annexin V-FITC apoptosis detection kit and Caspase-3/CPP32 Colorimetric Assay Kit were from BioVision (Mountain View, CA). RPMI-1640 and TRIZOL® reagent were purchased from Invitrogen (Argentina).

Cell culture

The doxorubicin-resistant (LBR-D160), vincristine-resistant (LBR-V160) and sensitive (LBR-) murine lymphoma cell lines were obtained previously in our laboratory.19 Human leukemic cell lines K562 (ATCC) and the resistant counterpart, a vincristine-selected cell line K562-Vinc were also used.20 Cell lines were grown in suspension cultures at 37°C in a 5% CO2 atmosphere using RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS).

Preparation of oHA

Oligomers ranging from HA4 tetrasaccharides to HA14 oligosaccharides were generated as described previously.21 Briefly, oligosaccharides were obtained after enzymatic digestion of LMW-HA (5 mg/ml) with bovine testicular hyaluronidase (Hase) employing 500 U/mg of HA incubating at 37°C for 24 hr. The reaction was stopped by boiling for 5 min. The size of oHA was determined by HPAEC-PAD.

Cytoplasmic extracts and western blot analysis

Cells were treated with 300 μg/ml of oHA, 200 μg/ml of native HA (HMW-HA), Hase control, anti-CD44 (KM81), anti-CD44 plus oHA, rat IgG2a (isotype control) or LY294002 for 2 hr. After this, cells were lysed with a hypotonic buffer (20 mM Tris pH 8.0, 150 mM NaCl, 100 mM NaF, 10% glycerol, 1% Nonidet P-40, 1 mM PMSF, 40 μg/ml leupeptin, 20 μg/ml aprotinin and 1 mM sodium orthovanadate) for 30 min at 4°C. After centrifugation, equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Sigma-Aldrich, St. Louis, MO). The membrane was blocked in Tris-buffered saline (TBS) containing 2% glycine and 3% non-fat dried milk, overnight at 4°C. The membrane was then incubated with specific antibodies to p-Akt, Akt, survivin or actin for 2 hr at 37°C followed by incubation with horseradish peroxidase-labeled secondary antibody for 1.5 hr at 37°C. The reaction was developed using a chemiluminescent detection system. Gel images obtained with a digital camera were subjected to densitometric analysis using Image Scion Software (Scion Corporation, USA).

PIP3 production

For the analysis of PIP3 production, cells were treated with 300 μg/ml oHA, 200 μg/ml HA or 10 μM LY294002 (PI3K inhibitor) for 30 min. PIP3 extraction was performed as described previously.22 Briefly, cells were incubated with cold 0.5 M TCA for 5 min, centrifuged and resuspended in 5% TCA/1 mM EDTA. After centrifugation, neutral lipids were extracted with methanol: chloroform (2:1) and acidic lipids by adding methanol:chloroform:12 M HCl (80:40:1). The extracts were centrifuged and chloroform plus 0.1 M HCl were added to the supernatant followed by centrifugation to separate organic and aqueous phases. The organic phase was dried in a vacuum dryer. Extracted lipids were spotted onto Immobilon membranes as described by Singleton et al.23 The dot membranes were blocked in TBS with 2% glycine and 3% nonfat dried milk, overnight at 4°C and then were probed with anti-PIP3 antibody, followed by horseradish peroxidase-labeled secondary antibody. Visualization of the immunoreactive areas was achieved using a chemiluminescent detection system and densitometric analysis was performed with Image Scion Software (Scion Corporation, USA).

Detection of apoptosis and caspase-3 activity

Cell lines (5 × 105 cells/ml) were either cultured in RPMI-1640 at 37°C in a 5% CO2 atmosphere for 24 hr in the presence of oHA (150 or 300 μg/ml), HA (200 μg/ml), Hase control, VCR (0.1 or 0.5 μM), DOX (0.1 or 0.5 μM) or cotreated with different doses of oHA plus VCR or DOX. Morphological features associated with apoptosis were analyzed by acridine orange and ethidium bromide staining.19 Briefly, cell pellets were resuspended in dye mixture (100 μg/ml acridine orange and 100 μg/ml ethidium bromide in phosphate-buffer saline) and visualized by fluorescence microscopy (Zeiss, Germany). A minimum of 200 cells were counted and the number of cells with fragmented nuclei, enlarged cytoplasm and condensed chromatin were determined. The percentage of apoptotic cells (apoptotic index) was calculated as: Apoptotic cells (%) = (total number of cells with apoptotic characteristics/total number of cells counted) × 100. Apoptosis induction was also evaluated by Annexin V staining method. Cells were resuspended in binding buffer and a mixture of Annexin V-FITC and prodipium iodide (PI) was added. Samples were analyzed using a Pas III flow cytometer (Partec, Germany) and data acquired were evaluated using WinMDI 2.8 software (Scripps Institute, La Jolla, USA). Late-stage apoptotic cells were labelled by positivity with Annexin V and Propidium iodide (right upper quadrant) and early-stage apoptotic cells were Annexin positive (right down quadrant). Percentage of apoptotic cells was assessed by adding the percentage of cells present in the 2 right quadrants. Caspase-3 activity was determined with a Caspase-3/CPP32 Colorimetric Assay Kit (Biovision, CA) according to the manufacturer's instructions. Briefly, after treatment (8 hr), cells were washed with cold PBS, resuspended in cell lysis buffer for 10 min, centrifuged at 10,000g for 5 min, and then 100 μg of protein was added to the reaction buffer containing 10 mM DTT. After the addition of DEVD-pNA substrate, the samples were incubated at 37°C for 1.5 hr and A405 was recorded. Fold-increase in caspase-3 activity was determined by comparing these results with the level of the uninduced control.

Drug efflux pump function

Intracellular accumulation of anthracyclines was carried out as previously described.24 Cells (5 × 105) were grown in drug-free medium for 24 hr prior to analysis, then stained for 40 min at 37°C with 200 mM daunorubicin (DNR) in the presence or absence of 8 mM cyclosporine A (CsA), 150 μg/ml oHA or 100 μg/ml anti-CD44 mAb (IM7.8.1). Stained cells were acquired and analyzed on a Pas III flow cytometer (Partec, Germany) and data were evaluated using WinMDI 2.8 software (Scripps Institute, La Jolla, USA). DNR fluorescence was collected through a 564–606 nm band-pass filter.

mRNA extraction and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Total mRNA from LBR-, LBR-D160 and LBR-V160 cell lines (5 × 106 cells) treated with 150 μg/ml oHA was isolated employing TRIZOL and RT-PCR was performed as described previously.24 cDNA was then amplified with specific primers for mdr-3 (sense 5′-TGGACCACATGACCAAGACAGGA-3′ and antisense 5′-ACCCTGTAGCCCCTTTCACTTGA-3′), and 30 PCR cycles (1 min at 94°C, 44 sec at 63°C, and 1 min at 72°C) were carried out, followed by a 10-min extension at 72°C. To evaluate the relative expression of mdr-3 genes, the β-actin gene was also amplified by 30 cycles (60 sec at 94°C, 60 sec at 53°C and 60 sec at 72°C) followed by a 10-min extension at 72°C, using the following pair of primers: sense 5′-ATGGATGACGATATCGCT-3′ and antisense 5′-ATGAGGTAGTCTGTCAGGT-3′. Serial dilutions were used for each cDNA to ensure that amplification occurred within the exponential range and that the reaction did not reach the plateau. PCR products were separated in 2% agarose gel, stained with ethidium bromide and visualized under UV light in a UV-transilluminator (Cole Palmer Instrumental, IL).

Statistical analysis

Apoptosis results were analyzed by one way-ANOVA and Tukey's test. Densitometric data from PIP3 production, western blot and mdr-3 mRNA were analyzed by one way-ANOVA and Dunnet's test. Analysis was performed using Prism software (Graph Pad, San Diego, CA, USA). p values < 0.05 or less were regarded as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

oHA induce apoptosis on multidrug resistant cell lines

To evaluate the ability of hyaluronan oligomers (oHA) to induce apoptosis in murine lymphoma resistant cell lines, cells were incubated with different doses of oHA and apoptosis was detected by both Annexin V staining and morphological features by acridine orange-ethidium bromide staining.

After treatment with 150 μg/ml oHA, we found higher apoptosis levels in LBR-D160 and LBR-V160 (13.5% ± 3.1%; 13.5% ± 3.4% respectively) than in LBR- (4.5% ± 2.2%) by acridine orange-ethidium bromide staining. When a concentration of 300 μg/ml oHA was tested, we observed enhanced apoptosis induction in LBR-D160 and LBR-V160 (18.9% ± 4.5%; 20.4% ± 3.5%, respectively) when compared with LBR- (7.4% ± 3.4%) (Fig. 1a). Since hyaluronan oligomers increased apoptosis in resistant cell lines, the effect of native HA on these cells was analyzed. However, native HA failed to induce apoptosis in both resistant and sensitive cell lines with doses of 200 μg/ml (Fig. 1a). As several HA concentrations (50–300 μg/ml) were assayed in previous studies and optimal results were obtained with 200 μg/ml, this HA dose was used for further experiments. oHA effect was considered specific since we used commercial HA prepared by fermentation of Streptococcus equi subsp. zooepidemicus bacterial strain in selecting production medium. HA was purified by repeated ultrafiltration, and specificity of the obtained product was tested by IR and UV spectrum (Contipro, Ústí nad Orlici, Czech Republic). To ruled out contamination from hyaluronidase, control was done using a solution containing hyaluronidase alone (incubated for 24 hr and then boiled at 90°C for 5 min).

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Figure 1. Apoptosis induction by oHA or native HA in LBR-, LBR-D160 and LBR-V160. (a) Analysis of morphological features by acridine orange-ethidium bromide staining treated with either 150 or 300 μg/ml oHA or 200 μg/ml native HA. Control was done using hyaluronidase tested alone to rule out possible effects of the enzyme on the cells. Percentage of apoptosis for each treatment was calculated by subtraction of spontaneous apoptosis from induced apoptosis (% apoptosis of treated cells − % apoptosis of untreated cells). Values are expressed as the means ± SD of apoptotic cells of 3 independent experiments. ***p < 0.001 vs. LBR-. (b) Annexin V staining of the 3 cell lines either untreated or treated with 300 μg/ml oHA. Percentage of apoptotic cells for each treatment was calculated by subtraction of apoptosis from untreated cells. One representative from 3 independent experiments is shown. (c) Caspase-3 activation was measured in total lysates of the cells treated with both doses of oHA or native HA for 8 hr. CsA (1 μg/ml) was used as positive control. Results represent the average of 3 independent experiments and are expressed as the means ± SD. * p < 0.05 vs. LBR-.

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Flow cytometry analysis of apoptosis by Annexin V and propidium iodide staining showed similar results to those obtained by morphological features (Fig. 1b).

To further confirm the apoptosis induction by oHA, the activity of the effector caspase-3 was investigated. As shown in Figure 1c, both doses of oHA (150 and 300 μg/ml) induced the activation of caspase-3. Similarly to the results obtained for apoptosis, native HA and Hase control failed to activate such caspase. Cyclosporine A (CsA) was used as positive control since we have previously demonstrated that this drug is able to induce caspase-3 activation in these cell lines.25

oHA modulate PI3K/Akt pathway and survivin expression

We have previously demonstrated that hyaluronan oligomers are able to induce cell death through inhibition of PI3K/Akt pathway in LBLa and LBLc murine lymphoma T cell lines.21 To investigate the effects of oHA and native HA on this pathway, PI3K activity was measured through the production of PIP3. As shown in Figure 2a, treatment with 300 μg/ml oHA significantly reduced PIP3 production in the 3 cell lines, comparable to inhibition levels obtained after treatment with the specific PI3K inhibitor, LY294002 (10 μM). Native HA showed an increase in PIP3 production only in LBR-.

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Figure 2. Effect of oHA or native HA on PI3K/Akt pathway and survivin expression. (a) Production of PIP3 was evaluated by dot blot. Densitometric analysis of the immunoreactive areas was performed and the results are expressed as the Index (treated cells/untreated cells). Bars represent means ± SD of 3 independent experiments. LY294002 was used as control of PI3K inhibition. (b) Expression of p-Akt and Akt was evaluated in the different cell lines (LBR-, LBR-D160 and LBR-V160) by western blot. Cytoplasmic extracts were prepared from untreated cells (Lane 1), Hase control (Lane 2) and treatment with 200 μg/ml HA (Lane 3) or 300 μg/ml oHA (Lane 4). Cotreatment with anti-CD44 plus oHA (Lane 5), anti-CD44 alone (Lane 6) and rat IgG2a isotype control (Lane 7) was performed. LY294002 (Lane 8) was used as positive control of PI3K inhibition. Expression of actin was used as loading control. Similar results were obtained in 3 independent experiments. Densitometric analysis of the bands was performed and the results are expressed as the index (treated cells/untreated cells), determined from 3 independent experiments. Bars represent means ± SD. (c) The effect of oHA and HA on survivin expression was evaluated by western blot followed by densitometric analysis of the bands. Expression of actin was used as loading control and the results are expressed as the index (treated cells/untreated cells), determined from 3 independent experiments. Bars represent means ± SD. * p < 0.05, ** p < 0.01 vs. untreated.

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We next evaluated the phosphorylation of Akt (p-Akt), the principal kinase downstream PI3K. Total extracts from the different cell lines either treated or not with oHA (300 μg/ml) or native HA (200 μg/ml) were prepared and western blot analyses were performed. As shown in Figure 2b, treatment with oHA decreased almost 30% the level of p-Akt in the 3 cell lines (LBR-: 25.1%, LBR-D160: 28.3% and LBR-V160: 33.5%), similar to the effect observed with LY294002. Cotreatment with anti-CD44 abolished the effect of oHA, being the expression of p-Akt similar to the untreated cells. Treatment with native HA showed a significant increase in p-Akt in LBR-, whereas this effect could not be observed in LBR-D160 and LBR-V160. Nevertheless, the biological effects of HA on these cell lines need further investigation. Besides, no changes in total Akt levels were found in the 3 cell lines.

Since survivin appeared to be elevated in some tumors, we also evaluated whether oHA modulated the expression of this survival factor in the 3 cell lines. As shown in Figure 2c, we found that oHA, but not HA, significantly downregulated survivin level only in LBR-V160 cell line (25.3%).

oHA sensitize MDR cells to vincristine

Since oHA increased apoptosis in resistant cell lines, we tested whether oHA were able to sensitize these cell lines to chemotherapeutic agents. Both LBR-D160 and LBR-V160 were incubated with oHA (150 and 300 μg/ml) in combination with either VCR (0.1 and 0.5 μM) or DOX (0.1 and 0.5 μM) for 24 hr. Apoptosis induction was evaluated by acridine orange and ethidium bromide staining showing that both doses of oHA were able to sensitize LBR-D160 to 0.5 μM VCR. As shown in Figure 3, cotreatment with oHA (150 and 300 μg/ml) and 0.5 μM VCR enhanced apoptosis levels (38.6% ± 2.7%; 34.0% ± 3.5%, respectively) when compared with that with oHA alone (18.9% ± 4.5%; 20.4% ± 3.5%, respectively). However, this effect was not observed with 0.1 μM VCR or any doses of DOX (data not shown). In LBR-V160, only treatment with 150 μg/ml oHA in combination with 0.5 μM VCR increased apoptosis induction when compared with oHA alone (30.9% ± 1.9% vs. 13.5% ± 3.4%). Similar data were obtained by Annexin V and propidium iodide staining (data not shown).

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Figure 3. Apoptosis induction by cotreatment with oHA and VCR in LBR-D160 and LBR-V160 cell lines. Detection of apoptosis by acridine orange and ethidium bromide staining. Cells were cultured in the presence of oHA (150 or 300 μg/ml), VCR (0.1 or 0.5 μM) or were cotreated with different doses of oHA plus VCR. Percentage of apoptotic cells was calculated by subtraction of spontaneous apoptosis from induced apoptosis. Results represent the average of 3 independent experiments and are expressed as the means ± SD. ns: nonsignificant, ** p < 0.01, *** p < 0.001.

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oHA modulate Pgp activity

Since one of the major mechanisms in MDR is the overexpression of transporter proteins such as Pgp, and having observed that oHA succeeded in sensitizing resistant cells to apoptosis, we investigated the role of oHA in Pgp modulation by flow cytometry analysis through DNR accumulation. As shown in Figure 4a, the sensitive cell line accumulated more DNR than the resistant cell lines. An increase in DNR fluorescence in both LBR-D160 and LBR-V160 was obtained with the specific Pgp inhibitor CsA, which was able to block drug efflux, thus confirming the presence of a functional pump in both resistant cell lines.

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Figure 4. Effect of oHA on Pgp modulation. (a) Flow cytometry analysis of daunorubicin efflux evaluated in LBR-, LBR-D160 and LBR-V160. The cells lines were incubated with DNR alone (first panel); DNR fluorescence was enhanced by CsA in both LBR-D160 and LBR-V160 cells as a result of drug efflux blockage (second panel). Modulation of Pgp in resistant cells treated with 150 μg/ml oHA (third panel), 100 μg/ml anti-CD44 plus 150 μg/ml oHA (fourth panel), or 100 μg/ml anti-CD44 (fifth panel). (b) Flow cytometry analysis of daunorubicin efflux in human leukemic cell lines K562 and K562-Vinc. The sensitive K562 and resistant K562-Vinc cells were incubated with DNR alone (first panel). DNR fluorescence was enhanced by CsA in K562-Vinc (second panel). Modulation of Pgp in resistant cells treated as shown in (a). Results are representative of 3 independent experiments. (c) Expression of mdr-3 was determined by RT-PCR on cells untreated or treated with 150 μg/ml oHA. β-actin cDNA was used as control of near equal amplification. Similar results were obtained at 2 and 15 hr. One representative from 3 independent experiments at 15 hr is shown.

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Treatment with oHA for 40 min presented enhanced intracellular fluorescence in LBR-D160 and LBR-V160 (92.6 and 51.0%, respectively) due to a blockage of DNR efflux (Fig. 4a panel 3). However, this effect was reversed after 24 hr of treatment (data not shown).

To evaluate whether the effect of oHA on Pgp function was mediated by interaction with CD44, resistant cells were treated with anti-CD44 mAb plus oHA. As shown in Figure 4a panel 4, the blocking effect of oHA on Pgp efflux was abolished in both resistant cell lines indicating that oHA could modulate Pgp activity through CD44. No changes in DNR efflux were observed with anti-CD44 alone (Fig. 4a panel 5).

We also analyzed oHA effect on Pgp activity in human leukemic cell lines K562 and vincristine resistant K562-Vinc. As shown in Figure 4b, oHA were also able to block drug efflux in K562-Vinc (35.2%). Moreover, when this human resistant cell line was coincubated with anti-CD44 mAb plus oHA, reversion of oHA effect was observed, indicating that CD44 was also involved in the effect of oHA on human Pgp function.

Since we have previously demonstrated that the resistant cell lines LBR-D160 and LBR-V160 express mdr-3,26 we examined mdr-3 expression after oHA treatment by RT-PCR. Despite the results observed in Pgp function, oHA failed to modulate mdr-3 expression in the resistant cell lines in the experimental conditions tested (Fig. 4c).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

MDR in hematological malignancies is the main reason of chemotherapy failure. Since multiple factors may contribute to chemoresistance, the impact of extracellular matrix components on this process has become of great interest.27, 28, 29 Hyaluronan appears to play an important role in tumor progression and metastasis as well as in MDR.

In this work we demonstrated that oHA- but not native HA- improve apoptosis in lymphoma resistant cell lines when compared with the sensitive line. Besides, we showed that oHA decreased PIP3 production and p-Akt expression in the three cell lines. This effect could be mediated by CD44 since inhibition of this receptor by anti-CD44 mAb abolished the effect of oHA on p-Akt. Our results are in agreement with previous studies demonstrating that oHA induces apoptosis by suppressing the PI3K/Akt cell survival pathway.15, 21 This pathway appears to be critical for survival of multidrug resistant cells. Indeed, aberrantly activated PI3K/Akt pathway increases antiapoptotic signals, overwhelms proapoptotic signals of antineoplasic drugs and confers drug resistance limiting success of chemotherapy.30 We have also demonstrated that PI3K/Akt inhibitors such as wortmannin and LY294002 were also able to induce higher apoptosis induction in LBR-D160 and LBR-V160 in comparison with LBR- (unpublished data). Thus, our findings suggest that oHA may act down regulating PI3K/Akt pathway, which plays a crucial role in chemoresistance in these resistant cell lines, explaining the higher apoptosis induction observed in LBR-D160 and LBR-V160 when compared with LBR-.

Alterations in proteins involved in cell death regulation such as survivin are also thought to play an important role in drug resistance. As survivin has been found overexpressed in many tumors such as lymphoma, melanoma and neuroblastoma, new strategies are being used to inhibit its expression as a way to restore programmed cell death.31, 32 Although oHA were able to decrease survivin expression in the resistant cell line LBR-V160, no effect was observed in LBR- and LBR-D160. Further research in other tumor models is necessary to confirm the ability of oHA to modulate survivin expression.

Our work demonstrated that oHA sensitize lymphoma resistant cell lines to vincristine. It has been previously reported that oHA are able to sensitize multidrug resistant breast tumor cells to antineoplasic agents, and that these effects are related with inhibition of PI3K and MAP kinase pathways.17 Our findings suggest that reversion of MDR in these lymphoma cell lines could be associated with the ability of oHA to decrease PI3K/Akt cell survival pathway as well as with inhibition of Pgp activity. Besides, we also showed that oHA failed to sensitize MDR cells lines to doxorubicin. This result is consistent with previous studies in which synergistic cytotoxicity was observed after treatment of glioma or ovarian cancer cells with PI3K/Akt inhibitor (LY294002) and antimicrotubule agents (such as vincristine or paclitaxel) but not with other antineoplasic drugs such as etoposide, cisplatin or BCNU.33, 34 Although the mechanisms involved in reversing MDR are not fully understood, we consider that the differences found between VCR (antimicrotubule agent) and DOX (topoisomerase II inhibitor) may be due to the fact that both anticancer drugs exert their effect through different mechanisms and could intercept PI3K/Akt pathway in a different way. From our results, we suggest that the combination of oHA with an antimicrotubule agent seems to be an effective therapeutic strategy to overcome resistance in lymphoma cell lines.

Since one of the most studied mechanisms of MDR is the overexpression of ATP-dependent efflux pumps such as Pgp, we also evaluated the ability of oHA to modulate Pgp. We found that oHA diminished Pgp efflux resulting in a cytoplasmic accumulation of cytotoxic drugs such as vincristine, and thus sensitizing resistant cell lines to this chemotherapeutic agent. Moreover, oHA were also able to block Pgp function in human leukemic resistant cell lines. Despite modulation of Pgp function by oHA, we did not observe any effect on mdr-3 expression. Previous results in breast carcinoma cell lines have shown that oHA decrease mdr-1 (Pgp) and mrp-2 (Mrp) expression, thus sensitizing these cell lines to doxorubicin.18 Our findings indicate that components of the extracellular matrix such as oHA may be able to act as Pgp inhibitors both in human and murine resistant cell lines and may be helpful to overcome MDR in Pgp-expressing cells from hematological malignancies.

We also showed that incubation with anti-CD44 mAb reverses the effects of oHA on Pgp activity. This result is particularly interesting because it suggests that oHA appears to be able to modulate Pgp function through its interaction with the CD44 receptor. Despite other reports describing that the effect of oHA is due to disruption of CD44-hyaluronan interactions in cancer cells,15, 17 we found that oHA exerts their effect by itself, probably mediated by CD44. These results suggest that this receptor could mediate the effect of oHA on MDR modulation. It has been recently described that the interaction between Pgp and CD44 modulates cell migration, invasion and metastasis. Moreover, it has been described that Pgp and CD44 coimmunoprecipitate and colocalize in the cell membrane.35 On the light of these findings, we suggest that oHA interact with CD44 leading to a conformational change in the CD44 receptor and a decrease in Pgp function. Besides, this would support the fact that we observed modulation in Pgp activity but not changes in its expression.

It has been recently reported that fragmented hyaluronan is able to induce the expression and function of Pgp in normal CD4 T cells. These results are not in agreement with those described by Misra et al.18 and ours. It has been suggested that the reason of these discrepancies could be the difference of the size of the oHA used.36 In fact, Tsujimura et al. used 36-mer oligomers while we, as Misra, used 4-14-mer oligomers.

The fact that oHA may have a different action depending on the cell type around the tumor is remarkable. It has been reported that oHA are able to activate lymphocytes or stimulate maturation of dendritic cells against tumors.36, 37 Besides, several studies (including ours) have demonstrated that oHA act sensitizing tumor cells to antineoplasic drugs by decreasing Pgp expression or inhibiting cell survival pathways. In our opinion, oHA may play a dual antitumor role since they both sensitize tumor cells to antineoplasic drugs and activate the immune response against the tumor.

In summary, in this work we demonstrated for the first time that oHA not only induce apoptosis in resistant lymphoma cell lines but are also able to sensitize these cells to the antineoplasic agent vincristine. Our findings suggest that reversion of MDR in this model could be related with the ability of oHA to decrease PI3K/Akt cell survival pathway as well as with inhibition of Pgp activity, indicating that both mechanisms could be essential for the survival of the resistant lymphoma cells lines. Thus, oHA could be able to modulate MDR and may be useful in combination with conventional agents in the treatment of hematological malignancies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We are grateful to Ms. Romina De León for technical assistance. We thank Dr. G. Lehne (University of Oslo, Norway) and Dr. Y. Assef (Instituto de Investigaciones Médicas Alfredo Lanari, Argentina) for kindly providing the K562-Vinc cell line and Dr. K. Mikecz (Rush University Medical Center, Chicago, USA) for KM81 (anti-CD44 mAb). Recombinant HMW and LMW-HA were provided by Farmatrade Argentina, Doxorubicin by Gador Pharmaceuticals Argentina, Vincristine sulfate by Filaxis Pharmaceuticals S.A. Argentina, and Daunorubicin by Laboratorios Varifarma S.A. Argentina.

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  2. Abstract
  3. Material and methods
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  5. Discussion
  6. Acknowledgements
  7. References
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